Accelerants for the Modification of Non-Natural Amino Acids and Non-Natural Amino Acid Polypeptides

ABSTRACT

Disclosed herein are accelerants for the formation of oxime-containing compounds from the reaction of a carbonyl-containing compound and a hydroxylamine-containing compound. The oxime-containing compound, the carbonyl-containing compound and the hydroxylamine-containing compound can each be a non-natural amino acid or a non-natural amino acid polypeptide. Also disclosed is the use of such accelerants to form oxime-containing compounds, the resulting oxime-containing compounds, and reaction mixtures containing such accelerants.

RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/734,589, entitled “Accelerants for the modification of non-naturalamino acids and non-natural amino acid polypeptides” filed on Nov. 8,2005.

FIELD OF THE INVENTION

Accelerants for the modification of molecules containing a carbonylmoiety, including non-natural amino acids and agents containing anon-natural amino acids.

BACKGROUND OF THE INVENTION

The ability to incorporate non-genetically encoded amino acids (i.e.,“non-natural amino acids”) into proteins permits the introduction ofchemical functional groups that could provide valuable alternatives tothe naturally-occurring functional groups, such as the epsilon —NH₂ oflysine, the sulfhydryl —SH of cysteine, the imino group of histidine,etc. Certain chemical functional groups are known to be inert to thefunctional groups found in the 20 common, genetically-encoded aminoacids but react cleanly and efficiently to form stable linkages withfunctional groups that can be incorporated onto non-natural amino acids.

Methods are now available to selectively introduce chemical functionalgroups that are not found in proteins, that are chemically inert to allof the functional groups found in the 20 common, genetically-encodedamino acids and that may be used to react efficiently and selectivelywith reagents comprising certain functional groups to form stablecovalent linkages.

SUMMARY OF THE INVENTION

Described herein are methods, compositions, techniques and strategiescomprising accelerants for the reaction of hydroxylamine-containingcompounds with carbonyl-containing compounds. The accelerants find usein the synthesis of oxime-containing compounds. The accelerants, in someembodiments, form bonds with the carbonyl-containing compounds, and assuch, these new compounds are more reactive withhydroxylamine-containing compounds. Described herein are chemicalcompounds that can modulate the reaction of hydroxylamine-containingcompounds with carbonyl-containing compounds. Also described herein arechemical compounds that can lower the activation barrier for thereaction of hydroxylamine-containing compounds with carbonyl-containingcompounds. Also described herein are chemical compounds that, whenincluded in a reaction comprising hydroxylamine-containing compounds andcarbonyl-containing compounds, increase the rate at whichoxime-containing compounds are formed. The hydroxylamine-, carbonyl-,and oxime-containing compounds can include non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides. The carbonyl-containing compounds include compoundscomprising an aromatic ketone moiety. Such compounds comprising anaromatic ketone moiety include amino acids and polypeptides. By way ofexample only, para-acetylphenylalanine or pAcF, is an amino acid thatcomprises an aromatic ketone moiety.

In one aspect are compounds that accelerate (referred to herein asaccelerants) the rate of reaction between hydroxylamine-containingcompounds with carbonyl-containing compounds to form oxime-containingcompounds.

In one embodiment, the hydroxylamine-containing compound is anon-natural amino acid, non-natural amino acid polypeptide or a modifiednon-natural amino acid polypeptide, and the carbonyl-containing compoundcomprises a desired functionality. In a further embodiment, theresulting oxime-containing compound comprises one of the aforementioneddesired groups (i.e., a desired functionality). In a related aspect arethe use of such compounds to accelerate the rate of reaction between ahydroxylamine-containing moiety on a non-natural amino acid, non-naturalamino acid polypeptide or a modified non-natural amino acid polypeptidewith a carbonyl-containing compound comprising a desired group (i.e., adesired functionality) to form an oxime-containing non-natural aminoacid, non-natural amino acid polypeptide or modified non-natural aminoacid polypeptide comprising a desired group. In another related aspectare reaction mixtures containing an accelerant, ahydroxylamine-containing non-natural amino acid, non-natural amino acidpolypeptide or modified non-natural amino acid polypeptide, and acarbonyl-containing compound comprising a desired group. In anotherrelated aspect are oxime-containing non-natural amino acids, non-naturalamino acid polypeptides or modified non-natural amino acid polypeptidescomprising a desired group, wherein such oxime-containing compounds areformed from the reaction of a hydroxylamine-containing non-natural aminoacid, non-natural amino acid polypeptide or modified non-natural aminoacid polypeptide with a carbonyl-containing compound comprising adesired group in the presence of an accelerant. In one embodiment, thecarbonyl group is not an aldehyde. In another embodiment, the carbonylgroup is an aromatic ketone.

In another embodiment, the carbonyl-containing compound is a non-naturalamino acid, non-natural amino acid polypeptide or a modified non-naturalamino acid polypeptide, and the hydroxylamine-containing compoundcomprises a desired functionality. In a further embodiment, theoxime-containing compound comprises one of the aforementioned groups. Ina related aspect are the use of such compounds to accelerate the rate ofreaction between a carbonyl-containing moiety on a non-natural aminoacid, non-natural amino acid polypeptide or a modified non-natural aminoacid polypeptide with a hydroxylamine-containing compound comprising adesired group to form an oxime-containing non-natural amino acid,non-natural amino acid polypeptide or modified non-natural amino acidpolypeptide comprising a desired group. In another related aspect arereaction mixtures containing an accelerant, a carbonyl-containingnon-natural amino acid, non-natural amino acid polypeptide or modifiednon-natural amino acid polypeptide, and a hydroxylamine-containingcompound comprising a desired group. In another related aspect areoxime-containing non-natural amino acids, non-natural amino acidpolypeptides or modified non-natural amino acid polypeptides comprisinga desired group, wherein such oxime-containing compounds are formed fromthe reaction of a carbonyl-containing non-natural amino acid,non-natural amino acid polypeptide or modified non-natural amino acidpolypeptide with a hydroxylamine-containing compound comprising adesired group in the presence of an accelerant. In one embodiment, thecarbonyl group is not an aldehyde. In another embodiment, the carbonylgroup is an aromatic ketone.

In a further aspect are methods for optimizing the reaction of acarbonyl-containing compound and a hydroxylamine-containing compound toform an oxime-containing compound by selection of at least oneappropriate accelerant. In one embodiment, such optimization comprisescomparing the yield of the oxime-containing compound in the presence ofdifferent accelerants, different molar ratios of accelerants, or acombination of the foregoing. In a further embodiment the yield of theoxime-containing compound is monitored by chromatography. In anotherembodiment, such optimization comprises comparing the amount ofside-products that result in the presence of different accelerants,different molar ratios of accelerants, or a combination of theforegoing. In a further embodiment the quantity of side products ismonitored by chromatography. In further embodiments, such optimizationincludes changing additional reaction conditions, including by way ofexample only pH and temperature. In one embodiment, the carbonyl groupis not an aldehyde. In another embodiment, the carbonyl group is anaromatic ketone.

In one aspect are non-natural amino acids based upon an oxime bond inwhich the oxime bond was formed in the presence of an accelerantdescribed herein. In further or additional embodiments, the non-naturalamino acid is incorporated into a polypeptide, that is, such embodimentsare non-natural amino acid polypeptides. In further or additionalembodiments, the non-natural amino acids are functionalized on theirsidechains such that their reaction with a derivatizing moleculegenerates an oxime bond formed in the presence of an accelerantdescribed herein. In further or additional embodiments are non-naturalamino acid polypeptides that can react with a derivatizing molecule,formed in the presence of an accelerant described herein (although sucha reaction may be less efficient in the absence of an accelerantdescribed herein), to generate an oxime-containing non-natural aminoacid polypeptide. In further or additional embodiments, the non-naturalamino acids are selected from amino acids having carbonyl, dicarbonyl orhydroxylamine sidechains. In further or additional embodiments, thenon-natural amino acids comprise carbonyl or dicarbonyl sidechains wherethe carbonyl or dicarbonyl is selected from a ketone or an aldehyde. Inanother embodiment are non-natural amino acids containing a functionalgroup that is capable of forming an oxime upon treatment with anappropriately functionalized co-reactant in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In afurther or additional embodiment, the non-natural amino acids resemble anatural amino acid in structure but contain one of the aforementionedfunctional groups. In another or further embodiment the non-naturalamino acids resemble phenylalanine or tyrosine (aromatic amino acids);while in a separate embodiment, the non-natural amino acids resemblealanine and leucine (hydrophobic amino acids). In one embodiment, thenon-natural amino acids have properties that are distinct from those ofthe natural amino acids. In one embodiment, such distinct properties arethe chemical reactivity of the sidechain, in a further embodiment thisdistinct chemical reactivity permits the sidechain of the non-naturalamino acid to undergo a reaction while being a unit of a polypeptideeven though the sidechains of the naturally-occurring amino acid unitsin the same polypeptide do not undergo the aforementioned reaction. In afurther embodiment, the sidechain of the non-natural amino acid has achemistry orthogonal to those of the naturally-occurring amino acids. Ina further embodiment, the sidechain of the non-natural amino acidcomprises an electrophile-containing moiety; in a further embodiment,the electrophile-containing moiety on the sidechain of the non-naturalamino acid can undergo nucleophilic attack to generate anoxime-derivatized protein in the presence of an accelerant describedherein (although such a reaction may be less efficient in the absence ofan accelerant described herein). In any of the aforementionedembodiments in this paragraph, the non-natural amino acid may exist as aseparate molecule or may be incorporated into a polypeptide of anylength; if the latter, then the polypeptide may further incorporatenaturally-occurring or non-natural amino acids. In one embodiment, thecarbonyl group is not an aldehyde. In another embodiment, the carbonylgroup is an aromatic ketone.

In another aspect are hydroxylamine-substituted molecules for theproduction of derivatized non-natural amino acid polypeptides based uponan oxime bond in the presence of an accelerant described herein(although such a reaction may be less efficient in the absence of anaccelerant described herein). In a further embodiment arehydroxylamine-substituted molecules used to derivatize carbonyl- ordicarbonyl-containing non-natural amino acid polypeptides via theformation of an oxime bond between the derivatizing molecule and thecarbonyl- or dicarbonyl-containing non-natural amino acid polypeptide inthe presence of an accelerant described herein (although such a reactionmay be less efficient in the absence of an accelerant described herein).In further embodiments the aforementioned carbonyl- ordicarbonyl-containing non-natural amino acid polypeptides areketo-containing non-natural amino acid polypeptides. In further oradditional embodiments, the carbonyl- or dicarbonyl-containingnon-natural amino acids comprise sidechains selected from a ketone or analdehyde. In further or additional embodiments, thehydroxylamine-substituted molecules comprise a desired functionality. Infurther or additional embodiments, the hydroxylamine-substitutedmolecules are hydroxylamine-substituted polyethylene glycol (PEG)molecules. In a further embodiment, the sidechain of the non-naturalamino acid has a chemistry orthogonal to those of thenaturally-occurring amino acids that allows the non-natural amino acidto react selectively with the hydroxylamine-substituted molecules in thepresence of an accelerant described herein (although such a reaction maybe less efficient in the absence of an accelerant described herein). Ina further embodiment, the sidechain of the non-natural amino acidcomprises an electrophile-containing moiety that reacts selectively withthe hydroxylamine-containing molecule in the presence of an accelerantdescribed herein (although such a reaction may be less efficient in theabsence of an accelerant described herein); in a further embodiment, theelectrophile-containing moiety on the sidechain of the non-natural aminoacid can undergo nucleophilic attack to generate an oxime-derivatizedprotein in the presence of an accelerant described herein (although sucha reaction may be less efficient in the absence of an accelerantdescribed herein). In a further aspect related to the embodimentsdescribed in this paragraph are the modified non-natural amino acidpolypeptides that result from the reaction of the derivatizing moleculewith the non-natural amino acid polypeptides in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). Furtherembodiments include any further modifications of the already modifiednon-natural amino acid polypeptides. In one embodiment, the carbonylgroup is not an aldehyde. In another embodiment, the carbonyl group isan aromatic ketone.

In another aspect are carbonyl- or dicarbonyl-substituted molecules forthe production of derivatized non-natural amino acid polypeptides basedupon an oxime bond, wherein the oxime bond is formed in the presence ofan accelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In afurther embodiment are carbonyl- or dicarbonyl-substituted moleculesused to derivatize hydroxylamine-containing non-natural amino acidpolypeptides via the formation of an oxime bond in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In afurther embodiment the carbonyl- or dicarbonyl-substituted molecules arealdehyde-substituted molecules or ketone-substituted moieties. Infurther embodiments, the carbonyl- or dicarbonyl-substituted moleculescomprise a desired functionality. In further or additional embodiments,the aldehyde-substituted molecules are aldehyde-substituted polyethyleneglycol (PEG) molecules. In further or additional embodiments, theketone-substituted molecules are ketone-substituted polyethylene glycol(PEG) molecules. In a further embodiment, the sidechain of thenon-natural amino acid has a chemistry orthogonal to those of thenaturally-occurring amino acids that allows the non-natural amino acidto react selectively with the carbonyl- or dicarbonyl-substitutedmolecules in the presence of an accelerant described herein (althoughsuch a reaction may be less efficient in the absence of an accelerantdescribed herein). In a further embodiment, the sidechain of thenon-natural amino acid comprises a moiety (e.g., hydroxylamine group)that reacts selectively with the carbonyl- or dicarbonyl-containingmolecule in the presence of an accelerant described herein (althoughsuch a reaction may be less efficient in the absence of an accelerantdescribed herein); in a further embodiment, the nucleophilic moiety onthe sidechain of the non-natural amino acid can undergo electrophilicattack to generate an oxime-derivatized protein in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In afurther aspect related to the embodiments described in this paragraphare the modified non-natural amino acid polypeptides that result fromthe reaction of the derivatizing molecule with the non-natural aminoacid polypeptides in the presence of an accelerant described herein(although such a reaction may be less efficient in the absence of anaccelerant described herein). Further embodiments include any furthermodifications of the already modified non-natural amino acidpolypeptides. In one embodiment, the carbonyl group is not an aldehyde.In another embodiment, the carbonyl group is an aromatic ketone.

In another aspect are mono-, bi- and multi-functional linkers for thegeneration of derivatized non-natural amino acid polypeptides based uponan oxime bond, wherein the oxime bond is formed in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In oneembodiment are molecular linkers (bi- and multi-functional) that can beused to connect carbonyl- or dicarbonyl-containing non-natural aminoacid polypeptides to other molecules in the presence of an accelerantdescribed herein (although such a reaction may be less efficient in theabsence of an accelerant described herein). In another embodiment aremolecular linkers (bi- and multi-functional) that can be used to connecthydroxylamine-containing non-natural amino acid polypeptides to othermolecules in the presence of an accelerant described herein (althoughsuch a reaction may be less efficient in the absence of an accelerantdescribed herein). In another embodiment the carbonyl- ordicarbonyl-containing non-natural amino acid polypeptides comprise aketone and/or an aldehyde sidechain. In an embodiment utilizing ahydroxylamine-containing non-natural amino acid polypeptide, themolecular linker contains a carbonyl or dicarbonyl group at one of itstermini; in further embodiments, the carbonyl or dicarbonyl group isselected from an aldehyde group or a ketone group. In further oradditional embodiments, the hydroxylamine-substituted linker moleculesare hydroxylamine-substituted polyethylene glycol (PEG) linkermolecules. In further or additional embodiments, the carbonyl- ordicarbonyl-substituted linker molecules are carbonyl- ordicarbonyl-substituted polyethylene glycol (PEG) linker molecules.Throughout, the phrase “other molecules” includes, by way of exampleonly, proteins, other polymers (branched and unbranched), smallmolecules, and groups also identified as a “desired functionality.” Infurther or additional embodiments, the hydroxylamine-containingmolecular linkers comprise the same or equivalent groups on all terminiso that upon reaction with a carbonyl- or dicarbonyl-containingnon-natural amino acid polypeptide in the presence of an accelerantdescribed herein (although such a reaction may be less efficient in theabsence of an accelerant described herein), the resulting product is thehomo-multimerization of the carbonyl- or dicarbonyl-containingnon-natural amino acid polypeptide. In further embodiments, thehomo-multimerization is a homo-dimerization. In further or additionalembodiments, the carbonyl- or dicarbonyl-containing molecular linkerscomprise the same or equivalent groups on all termini so that uponreaction with a hydroxylamine-containing non-natural amino acidpolypeptide in the presence of an accelerant described herein (althoughsuch a reaction may be less efficient in the absence of an accelerantdescribed herein), the resulting product is the homo-multimerization ofthe hydroxylamine-containing non-natural amino acid polypeptide. Infurther embodiments, the homo-multimerization is a homo-dimerization. Ina further embodiment, the sidechain of the non-natural amino acid has achemistry orthogonal to those of the naturally-occurring amino acidsthat allows the non-natural amino acid to react selectively with thehydroxylamine-substituted linker molecules in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In afurther embodiment, the sidechain of the non-natural amino acid has achemistry orthogonal to those of the naturally-occurring amino acidsthat allows the non-natural amino acid to react selectively with thecarbonyl- or dicarbonyl-substituted linker molecules in the presence ofan accelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In afurther embodiment, the sidechain of the non-natural amino acidcomprises an electrophile-containing moiety that reacts selectively withthe hydroxylamine-containing linker molecule in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein); in afurther embodiment, the electrophile-containing moiety on the sidechainof the non-natural amino acid can undergo nucleophilic attack by thehydroxylamine-containing linker molecule to generate anoxime-derivatized protein in the presence of an accelerant describedherein (although such a reaction may be less efficient in the absence ofan accelerant described herein). In a further aspect related to theembodiments described in this paragraph are the linked (modified)non-natural amino acid polypeptides that result from the reaction of thelinker molecule with the non-natural amino acid polypeptides. Furtherembodiments include any further modifications of the already linked(modified) non-natural amino acid polypeptides. In one embodiment, thecarbonyl group is not an aldehyde. In another embodiment, the carbonylgroup is an aromatic ketone.

In one aspect are methods to derivatize proteins via the condensation ofcarbonyl or dicarbonyl and hydroxylamine reactants in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein) to generatean oxime-based product. Included within this aspect are methods for thederivatization of proteins based upon the condensation of carbonyl- ordicarbonyl- and hydroxylamine-containing reactants to generate anoxime-derivatized protein adduct. In additional or further embodimentsare methods to derivatize keto-containing proteins withhydroxylamine-functionalized polyethylene glycol (PEG) molecules. In yetadditional or further aspects, the hydroxylamine-substituted moleculecan include proteins, other polymers (branched and unbranched), smallmolecules and groups also identified as a “desired functionality.” Inone embodiment, the carbonyl group is not an aldehyde. In anotherembodiment, the carbonyl group is an aromatic ketone.

In another aspect are methods for the chemical synthesis ofhydroxylamine-substituted molecules for the derivatization ofketo-substituted proteins in the presence of an accelerant describedherein (although such a reaction may be less efficient in the absence ofan accelerant described herein). In one embodiment, thehydroxylamine-substituted molecule can comprise peptides, other polymers(non-branched and branched) and small molecules. In one embodiment aremethods for the preparation of hydroxylamine-substituted moleculessuitable for the derivatization of carbonyl- or dicarbonyl-containingnon-natural amino acid polypeptides in the presence of an accelerantdescribed herein (although such a reaction may be less efficient in theabsence of an accelerant described herein), including by way of exampleonly, keto-containing non-natural amino acid polypeptides. In a furtheror additional embodiment, the non-natural amino acids are incorporatedsite-specifically during the in vivo translation of proteins. In afurther or additional embodiment, the hydroxylamine-substitutedmolecules allow for the site-specific derivatization of this carbonyl-or dicarbonyl-containing non-natural amino acid via nucleophilic attackof the carbonyl or dicarbonyl group to form an oxime-derivatizedpolypeptide in a site-specific fashion, wherein the oxime-derivatizedpolypeptide is formed in the presence of an accelerant described herein(although such a reaction may be less efficient in the absence of anaccelerant described herein). In a further or additional embodiment, themethod for the preparation of hydroxylamine-substituted moleculesprovides access to a wide variety of site-specifically derivatizedpolypeptides. In a further or additional embodiment are methods forsynthesizing hydroxylamine-functionalized polyethylene glycol (PEG)molecules.

In another aspect are methods for the chemical derivatization ofcarbonyl- or dicarbonyl-substituted non-natural amino acid polypeptides,in the presence of an accelerant described herein (although such areaction may be less efficient in the absence of an accelerant describedherein), using a hydroxylamine-containing bi-functional linker. In oneembodiment are methods for attaching a hydroxylamine-substituted linkerto a carbonyl- or dicarbonyl-substituted protein via a condensationreaction to generate an oxime bond in the presence of an accelerantdescribed herein (although such a reaction may be less efficient in theabsence of an accelerant described herein). In further or additionalembodiments, the carbonyl- or dicarbonyl-substituted non-natural aminoacid is a keto-substituted non-natural amino acid. In further oradditional embodiments, the non-natural amino acid polypeptides arederivatized site-specifically and/or with precise control ofthree-dimensional structure, using a hydroxylamine-containingbi-functional linker. In one embodiment, such methods are used to attachmolecular linkers (mono- bi- and multi-functional) to carbonyl- ordicarbonyl-containing (including by way of example keto-containing)non-natural amino acid polypeptides, wherein at least one of the linkertermini contains a hydroxylamine group which can link to the carbonyl-or dicarbonyl-containing non-natural amino acid polypeptides via anoxime bond in the presence of an accelerant described herein (althoughsuch a reaction may be less efficient in the absence of an accelerantdescribed herein). In a further or additional embodiment, these linkersare used to connect the carbonyl- or dicarbonyl-containing non-naturalamino acid polypeptides to other molecules, including by way of example,proteins, other polymers (branched and non-branched), small moleculesand groups also identified as a “desired functionality.” In oneembodiment, the carbonyl group is not an aldehyde. In anotherembodiment, the carbonyl group is an aromatic ketone.

In some embodiments, the non-natural amino acid polypeptide is linked toa water soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thepoly(ethylene glycol) molecule is a bifunctional polymer. In someembodiments, the bifunctional polymer is linked to a second polypeptide.In some embodiments, the second polypeptide is identical to the firstpolypeptide, in other embodiments, the second polypeptide is a differentpolypeptide. In some embodiments, the non-natural amino acid polypeptidecomprises at least two amino acids linked to a water soluble polymercomprising a poly(ethylene glycol) moiety.

In some embodiments, the non-natural amino acid polypeptide comprises asubstitution, addition or deletion that increases affinity of thenon-natural amino acid polypeptide for a receptor. In some embodiments,the non-natural amino acid polypeptide comprises a substitution,addition, or deletion that increases the stability of the non-naturalamino acid polypeptide. In some embodiments, the non-natural amino acidpolypeptide comprises a substitution, addition, or deletion thatincreases the aqueous solubility of the non-natural amino acidpolypeptide. In some embodiments, the non-natural amino acid polypeptidecomprises a substitution, addition, or deletion that increases thesolubility of the non-natural amino acid polypeptide produced in a hostcell. In some embodiments, the non-natural amino acid polypeptidecomprises a substitution, addition, or deletion that modulates proteaseresistance, serum half-life, immunogenicity, and/or expression relativeto the amino-acid polypeptide without the substitution, addition ordeletion.

In some embodiments, the non-natural amino acid polypeptide is anagonist, partial agonist, antagonist, partial antagonist, or inverseagonist. In some embodiments, the agonist, partial agonist, antagonist,partial antagonist, or inverse agonist comprises a non-natural aminoacid is linked to a water soluble polymer. In some embodiments, thewater polymer comprises a poly(ethylene glycol) moiety. In someembodiments, the polypeptide comprising a non-natural amino acid linkedto a water soluble polymer prevents dimerization of the correspondingreceptor. In some embodiments, the polypeptide comprising a non-naturalamino acid linked to a water soluble polymer modulates binding of thepolypeptide to a binding partner. In some embodiments, the polypeptidecomprising a non-natural amino acid linked to a water soluble polymermodulates one or more properties or activities of the polypeptide.

Also described herein are methods of making a non-natural amino acidpolypeptide linked to a water soluble polymer. In some embodiments, themethod comprises contacting an isolated polypeptide comprising anon-natural amino acid with a water soluble polymer comprising a moietythat reacts with the non-natural amino acid in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In someembodiments, the non-natural amino acid incorporated into is reactivetoward a water soluble polymer that is otherwise unreactive toward anyof the 20 common amino acids. In some embodiments, the water polymercomprises a poly(ethylene glycol) moiety. The molecular weight of thepolymer may be of a wide range, including but not limited to, betweenabout 100 Da and about 100,000 Da or more. The molecular weight of thepolymer may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of the polymer is between about 100 Da and about 50,000 Da. Insome embodiments, the molecular weight of the polymer is between about100 Da and about 40,000 Da. In some embodiments, the molecular weight ofthe polymer is between about 1,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 5,000Da and about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 10,000 Da and about 40,000 Da. In someembodiments, the poly(ethylene glycol) molecule is a branched polymer.The molecular weight of the branched chain PEG may be between about1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da,95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da,30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and1,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 1,000 Da and about 50,000 Da. In someembodiments, the molecular weight of the branched chain PEG is betweenabout 1,000 Da and about 40,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 5,000 Da and about40,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 5,000 Da and about 20,000 Da.

Also described herein are compositions comprising a polypeptidecomprising at least one of the non-natural amino acids described hereinand a pharmaceutically acceptable carrier. In some embodiments, thenon-natural amino acid is linked to a water soluble polymer. Alsodescribed herein are pharmaceutical compositions comprising apharmaceutically acceptable carrier and a polypeptide, wherein at leastone amino acid is substituted by a non-natural amino acid. In someembodiments, the non-natural amino acid comprises a saccharide moiety.In some embodiments, the water soluble polymer is linked to thepolypeptide via a saccharide moiety. Also described herein are prodrugsof the non-natural amino acids, non-natural amino acid polypeptides, andmodified non-natural amino acid polypeptides; further described hereinare compositions comprising such prodrugs and a pharmaceuticallyacceptable carrier. Also described herein are metabolites of thenon-natural amino acids, non-natural amino acid polypeptides, andmodified non-natural amino acid polypeptides; such metabolites may havea desired activity that complements or synergizes with the activity ofthe non-natural amino acids, non-natural amino acid polypeptides, andmodified non-natural amino acid polypeptides. Also described herein arethe use of the non-natural amino acids, non-natural amino acidpolypeptides, and modified non-natural amino acid polypeptides describedherein to provide a desired metabolite to an organism, including apatient in need of such metabolite.

Also described herein are libraries of the non-natural amino acidsdescribed herein or libraries of the non-natural amino acid polypeptidesdescribed herein, or libraries of the modified non-natural amino acidpolypeptides described herein, or combination libraries thereof, whereinthe members of the library comprise on oxime-linkage formed in thepresence of an accelerant described herein (although such a reaction maybe less efficient in the absence of an accelerant described herein).

Also described herein are methods for screening libraries describedherein for a desired activity, or for using the arrays to screen thelibraries described herein, or for other libraries of compounds and/orpolypeptides and/or polynucleotides for a desired activity. Alsodescribed herein is the use of such activity data from library screeningto develop and discover new therapeutic agents, as well as therapeuticagents themselves.

Also described herein are methods for accelerating the conjugation ofsmall molecules, including by way of example the conjugation of ahydroxylamine group on one reagent with a carbonyl group on anotherreagent, wherein neither reagent is a non-natural amino acid. In otherwords, the use of accelerants described herein is not limited to thefurther functionalization of non-natural amino acids and non-naturalamino acid polypeptides, but can also be used to facilitate theformation of oxime bonds between any two reagents. By way of exampleonly, this embodiment includes the use of accelerants in theformation/building of dynamic libraries from hydroxylamine-containingreagents and carbonyl-containing reagents. Of course, such dynamiclibraries can include non-natural amino acids, but such dynamiclibraries are not limited to the inclusion of non-natural amino acids.

Also described herein are methods of increasing therapeutic half-life,serum half-life or circulation time of a polypeptide that comprisesubstituting a non-natural amino acid for any one or more amino acids ina naturally occurring polypeptide, and/or adding a non-natural aminoacid into a naturally occurring polypeptide, and/or linking thepolypeptide to a water soluble polymer via an oxime bond formed in thepresence of an accelerant described herein (although such a reaction maybe less efficient in the absence of an accelerant described herein).

Also described herein are methods of treating a patient in need of suchtreatment with an effective amount of a pharmaceutical compositioncomprising a polypeptide comprising a non-natural amino acid comprisingan oxime bond formed in the presence of an accelerant described herein(although such a reaction may be less efficient in the absence of anaccelerant described herein) and a pharmaceutically acceptable carrier.In some embodiments, the non-natural amino acid is linked to a watersoluble polymer.

In any of the aforementioned aspects or embodiments, the use of anaccelerant includes the use of a single accelerant or multipleaccelerants. Further, in any of the aforementioned aspects orembodiments the molar ratio of accelerant to carbonyl-containingcompound includes values between about 0.5:1 to 5000:1, including by wayof example only 4000:1, 3000:1, 2000:1, 1000:1, 500:1, 400:1, 300:1,200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and 0.5:1. Further, inany of the aforementioned aspects or embodiments the molar ratio ofaccelerant to hydroxylamine-containing compound includes values betweenabout 0.5:1 to 5000:1, including by way of example only 4000:1, 3000:1,2000:1, 1000:1, 500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1,20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1,0.7:1, 0.6:1, and 0.5:1. Further, in any of the aforementioned aspectsor embodiments the accelerant includes compounds that can besubstantially removed in vacuo from the resulting oxime-containingcompound. Further, in any of the aforementioned aspects or embodimentsthe accelerant includes compounds containing an amine moiety, asemicarbazide moiety, a hydrazine, or a hydrazide moiety.

Further, in any of the aforementioned aspects or embodiments, theaccelerant is selected from the group consisting of bifunctionalaromatic amines, oxoamine derivatives, and compounds having thefollowing structures:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), C(═NH)—NH and SO, SO₂.

In a further embodiment, the accelerant is a bifunctional aromaticamine. In a further embodiment, the aromatic amine is selected from thegroup:

Bifuctional Aromatic Amines:

In a further embodiment, the accelerant is an oxoamine derivative. In afurther embodiment, the oxoamine derivative is selected from the group:

Oxoamine Derivatives:

In a further embodiment, the accelerant include compounds selected fromthe group consisting of:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), and C(═NH)—NH. Further, in any of theaforementioned aspects or embodiments, the accelerant is selected fromthe compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by wayof example any of compounds 6, 8, 10, 7, and 20 of FIG. 5. In any of theaforementioned aspects or embodiments, the accelerant includes an agentthat can form a hydrazone upon reaction with a carbonyl-containinggroup. Further, in any of the aforementioned aspects the accelerantactivity depends on the rate of reaction with the ketone moiety and thestability of the resulting intermediate. Further, in any of theaforementioned aspects or embodiments, the pH of the reaction mixturecomprising the accelerant, the carbonyl-containing compound and thehydroxylamine-containing compound is between about 2.0 and 10; betweenabout 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0;between about 4.0 and 6.0; between 3.0 and 10.0; between about 4.0 and10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; betweenabout 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0;between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0;about 5.5; about 6.0; about 6.5; and about 7.0. Note however, for any pHranges described herein, the phrase “between about” in reference to alow and high pH value means that the “about” applies to both the low andthe high pH value; by way of example only, “between about 3.0 and 10.0”is equivalent to “between about 3.0 and about 10.0.” Furthermore, unlessstated otherwise, for any range presented herein, in which “about” ispresented before a lower limit and not before an upper limit (or in thecase where “about” is placed before an upper limit and not a lowerlimit), then that is understood to mean that the word “about” appearsbefore both limits of the range.

Further, in any of the aforementioned aspects or embodiments, the term“accelerant” includes a compound having a least one of the followingproperties: (a) increase the rate of reaction between acarbonyl-containing compound and a hydroxylamine-containing compound toform an oxime-containing compound, where the increase in rate isrelative to the reaction in the absence of the accelerant; (b) lower theactivation energy of the reaction between a carbonyl-containing compoundand a hydroxylamine-containing compound to form an oxime-containingcompound, where the decrease in activation energy is relative to thereaction in the absence of the accelerant; (c) increase the yield of anoxime-containing compound from the reaction of a carbonyl-containingcompound with a hydroxylamine-containing compound, where the increase inyield is relative to the reaction in the absence of the accelerant; (d)lower the temperature at which a carbonyl-containing compound reactswith a hydroxylamine-containing compound to form an oxime-containingcompound, where the decrease in temperature is relative to the reactionin the absence of the accelerant; (e) decrease the time necessary toreact a carbonyl-containing compound with a hydroxylamine-containingcompound to form an oxime-containing compound, wherein the decrease intime is relative to the reaction in the absence of accelerant; (f)decrease the amount of reagents necessary to form an oxime-containingcompound, wherein the decrease in amount of reagents is relative to thereaction in the absence of accelerant; (g) decrease the side productsresulting from the reaction of a carbonyl-containing compound with ahydroxylamine-containing compound to form an oxime-containing compound,wherein the decrease in side products is relative to the reaction in theabsence of accelerant; (h) does not irreversibly destroy the tertiarystructure of a polypeptide undergoing an oxime-forming reaction in thepresence of an accelerant (excepting, of course, where the purpose ofthe reaction is to destroy such tertiary structure); (i) can beseparated from an oxime-containing compound in vacuo; and (j) modulatethe reaction of a carbonyl-containing compound with ahydroxylamine-containing compound. In a further embodiments, theaccelerant has at least two of the aforementioned properties, three ofthe aforementioned properties, four of the aforementioned properties,five of the aforementioned properties, six of the aforementionedproperties, seven of the aforementioned properties, eight of theaforementioned properties, nine of the aforementioned properties, or allof the aforementioned properties. In a further embodiment, theaccelerant has none of the aforementioned properties.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the methods and compositions described herein,which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the inventions described herein belong. Although anymethods, devices, and materials similar or equivalent to those describedherein can be used in the practice or testing of the inventionsdescribed herein, the preferred methods, devices and materials are nowdescribed.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH₂CH₂— and —CH₂CH₂CH₂CH₂—, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or allylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being a particularembodiment of the methods and compositions described herein. A “loweralkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The term “amino acid” refers to naturally occurring and non-naturalamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally encoded amino acids are the 20 common amino acids (alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline) and pyrrolysine and selenocysteine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, such as, homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups (such as, norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include but are not limited to various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

By “antibody fragment” is meant any form of an antibody other than thefull-length form. Antibody fragments herein include antibodies that aresmaller components that exist within full-length antibodies, andantibodies that have been engineered. Antibody fragments include but arenot limited to Fv, Fc, Fab, and (Fab′)₂, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, heavy chains, light chains, and variableregions, and alternative scaffold non-antibody molecules, bispecificantibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed.Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Anotherfunctional substructure is a single chain Fv (scFv), comprised of thevariable regions of the immunoglobulin heavy and light chain, covalentlyconnected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56,3055-3061). These small (Mr 25,000) proteins generally retainspecificity and affinity for antigen in a single polypeptide and canprovide a convenient building block for larger, antigen-specificmolecules. Unless specifically noted otherwise, statements and claimsthat use the term “antibody” or “antibodies” specifically includes“antibody fragment” and “antibody fragments.”

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (including but not limited to from 1 to 3 rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from one to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, aralkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“aralkyl” or “alkaryl” is meant to include those radicals in which anaryl group is attached to an alkyl group (including but not limited to,benzyl, phenethyl, pyridylmethyl and the like) including those alkylgroups in which a carbon atom (including but not limited to, a methylenegroup) has been replaced by, for example, an oxygen atom (including butnot limited to, phenoxymethyl, 2-pyridyloxymethyl,3-(1-naphthyloxy)propyl, and the like).

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789;all of which are incorporated by reference herein. A “multi-functionalpolymer” refers to a polymer comprising two or more discrete functionalgroups that are capable of reacting specifically with other moieties(including but not limited to, amino acid side groups) to form covalentor non-covalent linkages. A bi-functional polymer or multi-functionalpolymer may be any desired length or molecular weight, and may beselected to provide a particular desired spacing or conformation betweenone or more molecules linked to a compound and molecules it binds to orthe compound.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include butare not limited to any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drags, harddrugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes,lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the methods andcompositions described herein include, but are not limited to, drugs,prodrugs, radionuclides, imaging agents, polymers, antibiotics,fungicides, anti-viral agents, anti-inflammatory agents, anti-tumoragents, cardiovascular agents, anti-anxiety agents, hormones, growthfactors, steroidal agents, microbially derived toxins, and the like.

Cofolding, is used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, including but not limited to, by thelocal homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992)Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTalgorithm is typically performed with the “low complexity” filter turnedoff.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, less than about 0.01,and in another embodiment less than about 0.001.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of ordinary skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the deletion of an amino acid, addition of an amino acid, orsubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the methods and compositions described herein.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated, partially unsaturated and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. Additionally, the term encompasses bicyclicand tricyclic ring structures. Similarly, the term “heterocycloalkylene”by itself or as part of another substituent means a divalent radicalderived from heterocycloalkyl, and the term “cycloalkylene” by itself oras part of another substituent means a divalent radical derived fromcycloalkyl.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic, water misciblesolvents, phospholipids, or a combination of two or more such agents.Suitable chaotropes include, but are not limited to, urea, guanidine,and sodium thiocyanate. Useful detergents may include, but are notlimited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammnonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C₂-C₄ alkanols such as ethanol or isopropanol), orlower alkandiols (especially C₂-C₄ alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the methods andcompositions described herein may be naturally occurring phospholipidssuch as phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, and phosphatidylinositol or synthetic phospholipidderivatives or variants such as dihexanoylphosphatidylcholine ordiheptanoylphosphatidylcholine.

The term “desired functionality,” as used herein refers to any one orall of the following groups: a label; a dye; a polymer; a water-solublepolymer; a derivative of polyethylene glycol; a photocrosslinker; acytotoxic compound; a drug; an affinity label; a radionuclide; aderivative of biotin; a quantum dot; a nanotransmitter; aradiotransmitter; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin,a biomaterial; a nanoparticle; a spin label; a fluorophore, ametal-containing moiety; a radioactive moiety; a novel functional group;a group that covalently or noncovalently interacts with other molecules;a photocaged moiety; an actinic radiation excitable moiety; a ligand; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; a small molecule; aninhibitory ribonucleic acid, and any combination of the above.

The term “dicarbonyl” as used herein refers to a group containing atleast two moieties selected from the group consisting of —C(O)—, —S(O)—,—S(O)₂—, and —C(S)—, including, but not limited to, 1,2-dicarbonylgroups, a 1,3-dicarbonyl groups, and 1,4-dicarbonyl groups, and groupscontaining a least one ketone group, and/or at least one aldehydegroups, and/or at least one ester group, and/or at least one carboxylicacid group, and/or at least one thioester group. Such dicarbonyl groupsinclude diketones, ketoaldehydes, ketoacids, ketoesters, andketothioesters. In addition, such groups may be part of linear,branched, or cyclic molecules. The two moieties in the dicarbonyl groupmay be the same or different, and may include substituents that wouldproduce, by way of example only, an ester, a ketone, an aldehyde, athioester, or an amide, at either of the two moieties.

The term “effective amount” as used herein refers to that amount of the(modified) non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing the(modified) non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin degree, amount, potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem. When used in a patient, amounts effective for this use willdepend on the severity and course of the disease, disorder or condition,previous therapy, the patient's health status and response to the drugs,and the judgment of the treating physician.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, the same or different heteroatoms can also occupyeither or both of the chain termini (including but not limited to,alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,aminooxyalkylene, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages or bonds (the process of creating such alinkage or linker is referred to herein as linking/linked orcoupling/coupled, as well as other synonyms recognized by those in theart). Hydrolytically stable linkages means that the linkages aresubstantially stable in water and do not react with water at useful pHvalues, including but not limited to, under physiological conditions foran extended period of time, perhaps even indefinitely. Hydrolyticallyunstable or degradable linkages means that the linkages are degradablein water or in aqueous solutions, including for example, blood.Enzymatically unstable or degradable linkages means that the linkage canbe degraded by one or more enzymes. As understood in the art, PEG andrelated polymers may include degradable linkages in the polymer backboneor in the linker group between the polymer backbone and one or more ofthe terminal functional groups of the polymer molecule. For example,ester linkages formed by the reaction of PEG carboxylic acids oractivated PEG carboxylic acids with alcohol groups on a biologicallyactive agent generally hydrolyze under physiological conditions torelease the agent. Other hydrolytically degradable linkages include butare not limited to carbonate linkages; imine linkages resulted fromreaction of an amine and an aldehyde; phosphate ester linkages formed byreacting an alcohol with a phosphate group; hydrazone linkages which arereaction product of a hydrazide and an aldehyde; acetal linkages thatare the reaction product of an aldehyde and an alcohol; orthoesterlinkages that are the reaction product of a formate and an alcohol;peptide linkages formed by an amine group, including but not limited to,at an end of a polymer such as PEG, and a carboxyl group of a peptide;and oligonucleotide linkages formed by a phosphoramidite group,including but not limited to, at the end of a polymer, and a 5′ hydroxylgroup of an oligonucleotide.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into whichthe polypeptide has been secreted, including medium either before orafter a proliferation step. The term also may encompass buffers orreagents that contain host cell lysates, such as in the case where thepolypeptide is produced intracellularly and the host cells are lysed ordisrupted to release the polypeptide.

A “metabolite” of a (modified) non-natural amino acid polypeptidedisclosed herein is a derivative of that (modified) non-natural aminoacid polypeptide that is formed when the (modified) non-natural aminoacid polypeptide is metabolized. The term “active metabolite” refers toa biologically active derivative of a (modified) non-natural amino acidpolypeptide that is formed when the (modified) non-natural amino acidpolypeptide is metabolized. The term “metabolized” refers to the sum ofthe processes (including, but not limited to, hydrolysis reactions andreactions catalyzed by enzymes) by which a particular substance ischanged by an organism. Further information on metabolism may beobtained from The Pharmacological Basis of Therapeutics, 9th Edition,McGraw-Hill (1996). Metabolites of the (modified) non-natural amino acidpolypeptide disclosed herein can be identified either by administrationof (modified) non-natural amino acid polypeptide to a host and analysisof tissue samples from the host, or by incubation of (modified)non-natural amino acid polypeptide with hepatic cells in vitro andanalysis of the resulting compounds.

The term “modified,” as used herein refers to the presence of apost-translational modification on a polypeptide. The form “(modified)”term means that the polypeptides being discussed are optionallymodified, that is, the polypeptides under discussion can be modified orunmodified.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified polypeptiderelative to its non-modified form. Serum half-life is measured by takingblood samples at various time points after administration of thepolypeptide, and determining the concentration of that molecule in eachsample. Correlation of the serum concentration with time allowscalculation of the serum half-life. Increased serum half-life desirablyhas at least about two-fold, but a smaller increase may be useful, forexample where it enables a satisfactory dosing regimen or avoids a toxiceffect. In some embodiments, the increase is at least about three-fold,at least about five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of therapeuticallyeffective amount of a modified polypeptide, relative to its non-modifiedform. Therapeutic half-life is measured by measuring pharmacokineticand/or pharmacodynamic properties of the polypeptide at various timepoints after administration. Increased therapeutic half-life desirablyenables a particular beneficial dosing regimen, a particular beneficialtotal dose, or avoids an undesired effect. In some embodiments, theincreased therapeutic half-life results from increased potency,increased or decreased binding of the modified molecule to its target,increased or decreased breakdown of the molecule by enzymes such asproteases, or an increase or decrease in another parameter or mechanismof action of the non-modified molecule. As used herein, the term“non-eukaryote” refers to non-eukaryotic organisms. For example, anon-eukaryotic organism can belong to the Eubacteria (including but notlimited to, Escherichia coli, Thermus thermophilus, Bacillusstearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida etc.) phylogenetic domain, or the Archaea (includingbut not limited to, Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcusfuriosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogeneticdomain.

A “non-natural amino acid” refers to an amino acid that is not one ofthe 20 common amino acids or pyrrolysine or selenocysteine; other termsthat may be used synonymously with the term “non-natural amino acid” is“non-naturally encoded amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-natural amino acid”includes, but is not limited to, amino acids that occur naturally bymodification of a naturally encoded amino acid (including but notlimited to, the common amino acids or pyrrolysine and selenocysteine)but are not themselves incorporated into a growing polypeptide chain bythe translation complex. Examples of naturally-occurring amino acidsthat are not naturally-encoded include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, andO-phosphotyrosine.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Rossolim et al., Mol. Cell. Probes 8:91-98 (1994)).

“Oxidizing agent,” as used hereinwith respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Awide variety of oxidizing agents are suitable for use in the methods andcompositions described herein.

As used herein, the term “polyalkylene glycol” refers to polyethyleneglycol, polypropylene glycol, polybutylene glycol, and derivativesthereof. The term “polyalkylene glycol” encompasses both linear andbranched polymers and average molecular weights of between 0.1 kDa and100 kDa. Other exemplary embodiments are listed, for example, incommercial supplier catalogs, such as Shearwater Corporation's catalog“Polyethylene Glycol and Derivatives for Biomedical Applications”(2001).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-natural amino acid. As used herein, the terms encompass amino acidchains of any length, including full length proteins, wherein the aminoacid residues are linked by covalent peptide bonds.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

A “prodrug” refers to an agent that is converted into the parent drug invivo. Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. A pro-drug includes a pharmacologically inactive,or reduced-activity, derivative of an active drug. Prodrugs may bedesigned to modulate the amount of a drug or biologically activemolecule that reaches a desired site of action through the manipulationof the properties of a drug, such as physiochemical, biopharmaceutical,or pharmacokinetic properties. Prodrugs are converted into active drugwithin the body through enzymatic or non-enzymatic reactions. Prodrugsmay provide improved physiochemical properties such as bettersolubility, enhanced delivery characteristics, such as specificallytargeting a particular cell, tissue, organ or ligand, and improvedtherapeutic value of the drug.

In prophylactic applications, compositions containing the (modified)non-natural amino acid polypeptide are administered to a patientsusceptible to or otherwise at risk of a particular disease, disorder orcondition. Such an amount is defined to be a “prophylactically effectiveamount.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. It is considered well within theskill of the art for one to determine such prophylactically effectiveamounts by routine experimentation (e.g., a dose escalation clinicaltrial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein, includingphotolabile groups such as Nvoc and MeNvoc.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. A wide variety ofreducing agents are suitable for use in the methods and compositionsdescribed herein.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, or PNA, other nucleic acid mimcs, orcombinations thereof under conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m). 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (including butnot limited to, 10 to 50 nucleotides) and at least about 60° C. for longprobes (including but not limited to, greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For selective or specifichybridization, a positive signal may be at least two times background,optionally 10 times background hybridization. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C.,with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiment a human, who is the objectof treatment, observation or experiment.

The term “substantially purified” refers to a polypeptide that may besubstantially or essentially free of components that normally accompanyor interact with the protein as found in its naturally occurringenvironment, i.e. a native cell, or host cell in the case ofrecombinantly produced polypeptide. A polypeptide that may besubstantially free of cellular material includes preparations of proteinhaving less than about 30%, less than about 25%, less than about 20%,less than about 15%, less than about 10%, less than about 5%, less thanabout 4%, less than about 3%, less than about 2%, or less than about 1%(by dry weight) of contaminating protein. When the polypeptide orvariant thereof is recombinantly produced by the host cells, the proteinmay be present at about 30%, about 25%, about 20%, about 15%, about 10%,about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dryweight of the cells. When the polypeptide or variant thereof isrecombinantly produced by the host cells, the protein may be present inthe culture medium at about 5 g/L, about 4 g/L, about 3 g/L, about 2g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of thedry weight of the cells. Thus, “substantially purified” polypeptide asproduced by the methods described herein may have a purity level of atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, specifically, a purity level of atleast about 75%, 80%, 85%, and more specifically, a purity level of atleast about 90%, a purity level of at least about 95%, a purity level ofat least about 99% or greater as determined by appropriate methods suchas SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

The term “substituents” includes but is not limited to “non-interferingsubstituents.” “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₅-C₁₂ aralkyl, C₃-C₁₂cycloalkyl, C₄-C₁₂ cycloalkenyl, phenyl, substituted phenyl, toluoyl,xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₅-C₁₂ alkoxyaryl, C₅-C₁₂aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8, aryl, substitutedaryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substitutedheterocyclic radical, nitroalkyl, —NO₂, —CN, —NRC(O)—(C₁-C₁₀ alkyl),—C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkthioalkyl, —C(O)O—(C₁-C₁₀ alkyl), —OH,—SO₂, ═S, —COOH, —NR₂, carbonyl, —C(O)—(C₁-C₁₀ alkyl)-CF3, —C(O)—CF3,—C(O)NR2, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀ aryl), —C(O)—(C6-C₁₀ aryl),—(CH₂)_(m)—O—(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein each m is from 1 to 8,—C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂, —NRC(S)NR₂, salts thereof, andthe like. Each R group in the preceding list is independently selectedfrom the group consisting of H, alkyl or substituted alkyl, aryl orsubstituted aryl, or alkaryl. Where substituent groups are specified bytheir conventional chemical formulas, written from left to right, theyequally encompass the chemically identical substituents that wouldresult from writing the structure from right to left, for example,—CH₂O— is equivalent to —OCH₂—.

Substituents for alkyl and heteroalkyl radicals (including those groupsoften referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR, ═O, ═NR, N—OR, —NR₂, —SR, -halogen,—SiR₃, —OC(O)R, —C(O)R, —CO₂R, —CONR₂, —OC(O)NR₂, —NRC(O)R, —NR—C(O)NR₂,—NR(O)₂R, —NR—C(NR₂)═NR, —S(O)R, —S(O)₂R, —S(O)₂NR₂, —NRSO₂R, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such a radical. Each R group in the precedinglist is independently selected from the group consisting of hydrogen,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, including but not limited to, aryl substituted with 1-3 halogens,substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, oraralkyl groups. When two R groups are attached to the same nitrogenatom, they can be combined with the nitrogen atom to form a 5-, 6-, or7-membered ring. For example, —NR₂ is meant to include, but not belimited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussionof substituents, one of ordinary skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (including butnot limited to, —CF₃ and —CH₂CF₃) and acyl (including but not limitedto, —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for aryl and heteroaryl groups are varied and are selectedfrom, but are not limited to —OR, ═O, ═NR, ═N—OR, —NR₂, —SR, -halogen,—SiR₃, —OC(O)R, —C(O)R, —CO₂R, —CONR₂, —OC(O)NR₂, —NRC(O)R, —NR—C(O)NR₂,—NR(O)₂R, —NR—C(NR₂)═NR, —S(O)R, —S(O)₂R, —S(O)₂NR₂, —NRSO₂R, —CN, —NO₂,—R, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in anumber ranging from zero to the total number of open valences on thearomatic ring system; and where each R group in the preceding list isindependently selected from hydrogen, alkyl, heteroalkyl, aryl andheteroaryl.

In therapeutic applications, compositions containing the (modified)non-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto a polypeptide can result in changes including, but not limited to,increased or modulated serum half-life, or increased or modulatedtherapeutic half-life relative to the unmodified form, modulatedimmunogenicity, modulated physical association characteristics such asaggregation and multimer formation, altered receptor binding, alteredbinding to one or more binding partners, and altered receptordimerization or multimerization. The water soluble polymer may or maynot have its own biological activity. Suitable polymers include, but arenot limited to, polyethylene glycol, polyethylene glycolpropionaldehyde, mono C1-C10 alkoxy or aryloxy derivatives thereof(described in U.S. Pat. No. 5,252,714 which is incorporated by referenceherein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone,polyvinyl alcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include butare not limited to polyethylene glycol and serum albumin. In someembodiments, the water polymer comprises a poly(ethylene glycol) moiety.The molecular weight of the polymer may be of a wide range, includingbut not limited to, between about 100 Da and about 100,000 Da or more.The molecular weight of the polymer may be between about 100 Da andabout 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da,800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. Insome embodiments, the molecular weight of the polymer is between about100 Da and about 50,000 Da. In some embodiments, the molecular weight ofthe polymer is between about 100 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 1,000Da and about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and about 40,000 Da. In some embodiments, the poly(ethylene glycol)molecule is a branched polymer. The molecular weight of the branchedchain PEG may be between about 1,000 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 1,000 Da and about50,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 1,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the branched chain PEG is betweenabout 5,000 Da and about 40,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 5,000 Da and about20,000 Da.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

Compounds (including, but not limited to non-natural amino acids,(modified) non-natural amino acid polypeptides and reagents forproducing either of the aforementioned compounds) presented hereininclude isotopically-labelled compounds, which are identical to thoserecited in the various formulas and structures presented herein, but forthe fact that one or more atoms are replaced by an atom having an atomicmass or mass number different from the atomic mass or mass numberusually found in nature. Examples of isotopes that can be incorporatedinto the present compounds include isotopes of hydrogen, carbon,nitrogen, oxygen, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certain isotopically-labelledcompounds described herein, for example those into which radioactiveisotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/orsubstrate tissue distribution assays. Further, substitution withisotopes such as deuterium, i.e., ²H, can afford certain therapeuticadvantages resulting from greater metabolic stability, for exampleincreased in vivo half-life or reduced dosage requirements.

Some of the compounds herein (including, but not limited to non-naturalamino acids, (modified) non-natural amino acid polypeptides and reagentsfor producing either of the aforementioned compounds) have asymmetriccarbon atoms and can therefore exist as enantiomers or diastereomers.Diasteromeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods known, for example, by chromatography and/or fractionalcrystallization. Enantiomers can be separated by converting theenantiomeric mixture into a diastereomeric mixture by reaction with anappropriate optically active compound (e.g., alcohol), separating thediastereomers and converting (e.g., hydrolyzing) the individualdiastereomers to the corresponding pure enantiomers. All such isomers,including diastereomers, enantiomers, and mixtures thereof areconsidered as part of the compositions described herein.

In additional or further embodiments, the compounds described herein(including, but not limited to non-natural amino acids, (modified)non-natural amino acid polypeptides and reagents for producing either ofthe aforementioned compounds) are used in the form of pro-drugs. Inadditional or further embodiments, the compounds described herein(including, but not limited to non-natural amino acids, (modified)non-natural amino acid polypeptides and reagents for producing either ofthe aforementioned compounds) are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In a further oradditional embodiments are active metabolites of non-natural amino acidsand (modified) non-natural amino acid polypeptides.

The methods and formulations described herein include the use ofN-oxides, crystalline forms (also known as polymorphs), orpharmaceutically acceptable salts of non-natural amino acids and(modified) non-natural amino acid polypeptides. In some situations,non-natural amino acids and (modified) non-natural amino acidpolypeptides may exist as tautomers. All tautomers are included withinthe scope of the non-natural amino acids and (modified) non-naturalamino acid polypeptides presented herein. In addition, the non-naturalamino acids and (modified) non-natural amino acid polypeptides describedherein can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. The solvated forms of the non-natural amino acids and (modified)non-natural amino acid polypeptides presented herein are also consideredto be disclosed herein.

Those of ordinary skill in the art will recognize that some of thecompounds herein (including, but not limited to non-natural amino acids,(modified) non-natural amino acid polypeptides and reagents forproducing either of the aforementioned compounds) can exist in severaltautomeric forms. All such tautomeric forms are considered as part ofthe compositions described herein. Also, for example all enol-keto formsof any compounds (including, but not limited to non-natural amino acids,(modified) non-natural amino acid polypeptides and reagents forproducing either of the aforementioned compounds) herein are consideredas part of the compositions described herein.

Some of the compounds herein (including, but not limited to non-naturalamino acids, (modified) non-natural amino acid polypeptides and reagentsfor producing either of the aforementioned compounds) are acidic and mayform a salt with a pharmaceutically acceptable cation. Some of thecompounds herein (including, but not limited to non-natural amino acids,(modified) non-natural amino acid polypeptides and reagents forproducing either of the aforementioned compounds) can be basic andaccordingly, may form a salt with a pharmaceutically acceptable anion.All such salts, including di-salts are within the scope of thecompositions described herein and they can be prepared by conventionalmethods. For example, salts can be prepared by contacting the acidic andbasic entities, in either an aqueous, non-aqueous or partially aqueousmedium. The salts are recovered by using at least one of the followingtechniques: filtration, precipitation with a non-solvent followed byfiltration, evaporation of the solvent, or, in the case of aqueoussolutions, lyophilization.

Salts, for example, include: (1) acid addition salts, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like; or formed with organicacids such as acetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid,malonic acid, succinic acid, malic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; (2) salts formed when anacidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base. Acceptable organicbases include ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like. Acceptable inorganicbases include aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like.

It should be understood that a reference to a salt includes the solventaddition forms or crystal forms thereof, particularly solvates orpolymorphs. Solvates contain either stoichiometric or non-stoichiometricamounts of a solvent, and are often formed during the process ofcrystallization. Hydrates are formed when the solvent is water, oralcoholates are formed when the solvent is alcohol. Polymorphs includethe different crystal packing arrangements of the same elementalcomposition of a compound. Polymorphs usually have different X-raydiffraction patterns, infrared spectra, melting points, density,hardness, crystal shape, optical and electrical properties, stability,and solubility. Various factors such as the recrystallization solvent,rate of crystallization, and storage temperature may cause a singlecrystal form to dominate.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinventions. As one example, the following patent applications aredisclosed in their entirety: 60/638,418; 60/696,210; 60/638,527;60/696,302; 60/639,195; 60/696,068; 60/755,338; 60/755,711; 60/755,018;60/743,041; 60/743,040; 60/734,589; Ser. Nos. 11/313,956; 11/313,306;and 11/313,305. The publications discussed herein are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors described herein are not entitled to antedate such disclosureby virtue of prior invention or for any other reason.

BRIEF DESCRIPTION OF THE FIGURES

A better understanding of the features and advantages of the presentmethods and compositions may be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of our methods, compositions, devices and apparatuses areutilized, and the accompanying drawings of which:

FIG. 1 presents a schematic representation of the relationship ofcertain aspects of the methods, compositions, strategies and techniquesdescribed herein.

FIG. 2 presents a non-limiting example of a SDS-PAGE analysis of onestep dimerization reactions of scFv 108 using 2 K homobifunctionalhydroxylamine PEG linker with different molar ratios: 1)scFv:linker=1.6:1, with acetic hydrazide; 2) scFv:linker=2:1, withacetic hydrazide; 3) scFv:linker=2.4:1, with acetic hydrazide; 4)scFv:linker=2:1, without acetic hydrazide; 5) scFv:linker=2:1, withacetic hydrazide without PEG linker.

FIG. 3 presents a non-limiting example of a SDS-PAGE analysis ofscFv-pAcF and 30 K mono hydroxylamine PEG conjugation 1) the standard of100% starting scFv-pAcF; 2) the standard of 20% starting scFv-pAcF; 3)the standard of 10% starting scFv-pAcF; 4) scFv:PEG=1:3 with 20 mMacetic hydrazide; 5) scFv:PEG=1:3 without acetic hydrazide; 6)scFv:PEG=1:5 with 20 mM acetic hydrazide; 7) scFv:PEG=1:5 without acetichydrazide.

FIG. 4 presents a non-limiting example of a SDS-PAGE analysis ofscFv-pAcF and 30 K mono hydroxylamine PEG conjugation with differentconcentration of acetic hydrazide. 1) scFv-pAcF:PEG=1:2, 5 mM acetichydrazide; 2) scFv-pAcF:PEG=1:2, 20 mM acetic hydrazide; 3)scFv-pAcF:PEG 1:2, 80 mM acetic hydrazide; 4) scFv-pAcF:PEG=1:5, noacetic hydrazide; 5) the standard of 10% scFv-pAcF; 6) the standard of20% scFv-pAcF; 7) the standard of 100% scFv-pAcF.

FIG. 5 presents non-limiting examples of accelerants that can be used inthe methods, reactions and syntheses described herein.

FIG. 6 presents a non-limiting example of an SDS-PAGE analysis comparingthe formation of oxime in the presence of different accelerants; thelane number corresponds to the accelerant number in FIG. 5 and the lastlane is a control reaction without accelerant.

FIG. 7 presents a non-limiting example of an SDS-PAGE analysis of theconjugation of hGH-pAcF with 30 K monohydroxylamine PEG with accelerants7 and 20:1) hGH-pAcF:PEG=1:2 with accelerant 7; 2) hGH-pAcF PEG=1:2 withaccelerant 20; 3) hGH-pAcF:PEG=1:2 no accelerant; 4) hGH-pAcF:PEG=1:5 noaccelerant.

FIG. 8 presents a non-limiting example of an LCMS analysis of hGHincubated with different concentrations of accelerant acetic hydrazide:A) total LCMS trace; B) Mass spectrum of hGH without accelerant; C) Massspectrum of hGH with 200 mM accelerant acetic hydrazide.

FIG. 9 presents non-limiting examples of accelerants that can be used inthe methods, reactions and syntheses described herein.

FIG. 10 a presents a non-limiting reaction of a model ketone with amodel hydroxylamine in the presence of an accelerant to form a modeloxime; FIG. 10 b presents non-limiting examples of accelerants that canbe used in the methods, reactions and syntheses described herein.

FIG. 11 presents a non-limiting set of oxime yields for a model reactionconducted in the absence and in the presence of various accelerantsdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Saccharomyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-natural amino acids to proteins invivo. A number of new amino acids with novel chemical, physical orbiological properties, including photoaffinity labels andphotoisomerizable amino acids, photocrosslinking amino acids (see, e.g.,Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:11020-11024;and Chin, J. W., et al. (2002) J. Am. Chem. Soc. 124:9026-27); ketoamino acids, and glycosylated amino acids have been incorporatedefficiently and with high fidelity into proteins in E. coli and in yeastin response to the amber codon, TAG, using this methodology. See, e.g.,S. W. Chin et al., (2002), Journal of the American Chemical Society124:9026-9027 (incorporated by reference in its entirety); J. W. Chin, &P. G. Schultz, (2002), Chem Bio Chem 3(11):1135-1137 (incorporated byreference in its entirety); J. W. Chin, et al, (2002), PNAS UnitedStates of America 99:11020-11024 (incorporated by reference in itsentirety); and, L. Wang, & P. G. Schultz, (2002), Chem. Comm. 1:1-11(incorporated by reference in its entirety). These studies havedemonstrated that it is possible to selectively and routinely introducechemical functional groups that are not found in proteins, that arechemically inert to all of the functional groups found in the 20 common,genetically-encoded amino acids (i.e., “natural” amino acids) and thatmay be used to react efficiently and selectively to form stable covalentlinkages.

Chemical functional groups not found in the natural amino acids includecarbonyl groups, such as ketones and aldehydes, and hydroxylaminegroups. A hydroxylamine moiety reacts with a carbonyl group such asketone and aldehyde to form a relatively stable oxime; this pairing(hydroxylamine with a carbonyl group) thus provides a means for furtherfunctionalizing non-natural amino acid polypeptides. One non-limitingexample of such a pairing is shown below:

For example, when either a hydroxylamine moiety or a carbonyl group isincorporated into a non-natural amino acid polypeptide and reacted witha reagent that contains that other member of the pair, the non-naturalamino acid polypeptide can be functionalized with the reagent via theformation of an oxime group. Though compatible for proteinfunctionalization reactions, the standard oxime formation reaction maybe made more efficient which would allow, for example, for the use oflower amounts of reactants and reduce the time to reaction completion.Therefore, the development of an accelerant is highly desirable.

II. Overview

FIG. 1 is one embodiment of the compositions, methods and techniquesthat are described herein. A carbonyl-containing compound is selectedfor reaction with a hydroxylamine-containing compound to form anoxime-containing compound. The carbonyl-containing compound includesnon-natural amino acids, polypeptides, oligonucleotides, polymers(including by way of example only polyethylene glycol), reagents, linkergroups, groups comprising further functionality, and combinationsthereof, the present disclosure provides numerous examples of groupscomprising further functionality. The hydroxylamine-containing compoundincludes non-natural amino acids, polypeptides, oligonucleotides,polymers (including by way of example only polyethylene glycol),reagents, linker groups, groups comprising further functionality, andcombinations thereof; the present disclosure provides numerous examplesof groups comprising further functionality. The oxime-containingcompound includes non-natural amino acids, polypeptides,oligonucleotides, polymers (including by way of example onlypolyethylene glycol), reagents, linker groups, groups comprising furtherfunctionality, and combinations thereof; the present disclosure providesnumerous examples of groups comprising further functionality. To thereaction mixture of the hydroxylamine-containing compound and thecarbonyl-containing compound is added an accelerant, wherein theaccelerant has at least one of the following properties: (a) increasethe rate of reaction between a carbonyl-containing compound and ahydroxylamine-containing compound to form an oxime-containing compound,where the increase in rate is relative to the reaction in the absence ofthe accelerant; (b) lower the activation energy of the reaction betweena carbonyl-containing compound and a hydroxylamine-containing compoundto form an oxime-containing compound, where the decrease in activationenergy is relative to the reaction in the absence of the accelerant; (c)increase the yield of an oxime-containing compound from the reaction ofa carbonyl-containing compound with a hydroxylamine-containing compound,where the increase in yield is relative to the reaction in the absenceof the accelerant; (d) lower the temperature at which acarbonyl-containing compound reacts with a hydroxylamine-containingcompound to form an oxime-containing compound, where the decrease intemperature is relative to the reaction in the absence of theaccelerant; (e) decrease the time necessary to react acarbonyl-containing compound with a hydroxylamine-containing compound toform an oxime-containing compound, wherein the decrease in time isrelative to the reaction in the absence of accelerant; (f) decrease theamount of reagents necessary to form an oxime group on a non-naturalamino acid polypeptide, wherein the decrease in amount of reagents isrelative to the reaction in the absence of accelerant; (g) decrease theside products resulting from the reaction of a carbonyl-containingcompound with a hydroxylamine-containing compound to form anoxime-containing compound, wherein the decrease in side products isrelative to the reaction in the absence of accelerant; (h) does notirreversibly destroy the tertiary structure of a polypeptide undergoingan oxime-forming reaction in the presence of an accelerant (excepting,of course, where the purpose of the reaction is to destroy such tertiarystructure); (i) can be separated from an oxime-containing compound invacuo; and (j) modulate the reaction of a carbonyl-containing compoundwith a hydroxylamine-containing compound. In a further embodiment, theaccelerant has none of the aforementioned properties. Optionally, avariety of accelerants described herein are tested and an accelerant isselected based on its possession of at least one of the aforementionedproperties. Optionally, the reaction characteristics (e.g., yield ofoxime-containing compound) can be further optimized by at least one ofthe following: (a) varying the amount of accelerant, (b) varying theamount of carbonyl-containing compound, (c) varying the amount ofhydroxylamine-containing compound, (d) varying the temperature of thereaction, (e) varying the pH of the reaction, and (f) varying thesolvent in the reaction mixture. Optionally, additional accelerants aretested at an optimized reaction condition, or the selection andoptimization steps are reversed, or the selection and optimization stepsare repeated in an iterative fashion. The carbonyl-containing compoundand the hydroxylamine-containing compound are reacted in the presence ofthe accelerant to form the oxime-containing compound. Optionally theprogress of the reaction is monitored by a detection means, including byway of example only, chromatography. The oxime-containing compoundresulting from the reaction of a carbonyl-containing compound and ahydroxylamine-containing compound in the presence of an accelerant canbe optionally isolated, purified and characterized. The accelerant canbe removed from the oxime-containing compound by a variety of methods,including by way of example only filtration, in vacuo techniques,chromatography, membrane-based bioseparation, electrophoresis,precipitation of the oxime-containing compound, distillation, or acombination thereof. Thus, in one embodiment described herein, theaccelerants can be removed in vacuo from the oxime-containing material;however, in other embodiments described herein, the accelerants can beremoved using any (or any combination) of the aforementioned methods.Isolation and purification signifies the removal of at least somenon-oxime containing compound from the materials in the reactionmixture.

At one level, described herein are the tools (methods, compositions,techniques) for creating and using a polypeptide comprising at least onenon-natural amino acid or modified non-natural amino acid with an oximegroup formed in the presence of an accelerant (although such a reactionmay be less efficient in the absence of an accelerant described herein).Such non-natural amino acids may contain further functionality,including but not limited to, a desired functionality.

Also described herein are non-natural amino acids that have or can bemodified to contain an oxime moiety formed in the presence of anaccelerant (although such a reaction may be less efficient in theabsence of an accelerant described herein). Included with this aspectare methods for producing, purifying, characterizing and using suchnon-natural amino acids. In another aspect described herein are methods,strategies and techniques for incorporating at least one suchnon-natural amino acids into a polypeptide. Also included with thisaspect are methods for producing, purifying, characterizing and usingsuch polypeptides containing at least one such non-natural amino acids.Also included with this aspect are compositions of and methods forproducing, purifying, characterizing and using polynucleotides(including DNA and RNA) that can be used to produce, at least in part, apolypeptide containing at least one non-natural amino acid that canreact, in the presence of an accelerant (although such a reaction may beless efficient in the absence of an accelerant described herein) to forman oxime-containing non-natural amino acid polypeptide, including such apolypeptide that has been modified. Also included with this aspect arecompositions of and methods for producing, purifying, characterizing andusing cells that can express such polynucleotides that can be used toproduce, at least in part, a polypeptide containing at least onenon-natural amino acid.

Also included within the scope of the methods, compositions, strategiesand techniques described herein are accelerants for reacting a reagentwith a non-natural amino acid (containing a carbonyl or dicarbonylgroup, hydroxylamine group, or protected forms thereof) that is part ofa polypeptide so as to produce any of the aforementionedpost-translational modifications. In general, the resultingpost-translationally modified non-natural amino acid polypeptide willcontain at least one oxime group; the resulting modifiedoxime-containing non-natural amino acid polypeptide may undergosubsequent modification reactions. Also included with this aspect aremethods for selecting, producing, optimizing, purifying, characterizingand using such accelerants that can be used with any suchpost-translational modifications of such non-natural amino acid(s).

The non-natural amino acid containing polypeptide can contain at leastone, at least two, at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, or ten or morenon-natural amino acids containing an oxime group (or protected ormasked forms thereof), wherein at least one oxime group was produced inthe presence of the accelerants described herein, and further, such anoxime-containing non-natural amino acid polypeptide may optionallycontain at least non-natural amino acid polypeptide containing onecarbonyl or dicarbonyl group, hydroxylamine group, or protected formsthereof. The non-natural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more different sites in the protein that comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,or more different non-natural amino acids. In certain embodiments, atleast one, but fewer than all, of a particular amino acid present in anaturally occurring version of the protein is substituted with anon-natural amino acid.

The non-natural amino acid methods and compositions described hereinprovides conjugates of substances having a wide variety of functionalgroups (provided that at least one conjugate is chemically linked to anon-natural amino acid via an oxime group formed in the presence of theaccelerants described herein), substituents or moieties, with othersubstances including but not limited to a desired functionality.

In another aspect of the compositions, methods, techniques andstrategies described herein are methods for studying or using any of theaforementioned (modified) non-natural amino acid polypeptides. Includedwithin this aspect, by way of example only, are therapeutic, diagnostic,assay-based, industrial, cosmetic, plant biology, environmental,energy-production, and/or military uses which would benefit from apolypeptide comprising a (modified) non-natural amino acid polypeptideor protein.

III. Post-Translational Modifications of Non-Natural Amino AcidComponents of a Polypeptide in the Presence of at Least One Accelerant

Methods, compositions, techniques and strategies have been developed tosite-specifically incorporate non-natural amino acids during the in vivotranslation of proteins. By incorporating a non-natural amino acid witha sidechain chemistry that is orthogonal to those of thenaturally-occurring amino acids, this technology makes possible thesite-specific derivatization of recombinant proteins. As a result, amajor advantage of the methods, compositions, techniques and strategiesdescribed herein is that derivatized proteins can now be prepared asdefined homogeneous products. However, the methods, compositions,reaction mixtures, techniques and strategies described herein involvingan accelerant are not limited to non-natural amino acid polypeptidesformed by in vivo protein translation techniques, but includesnon-natural amino acid polypeptides formed by any technique, includingby way of example only expressed protein ligation, chemical synthesis,ribozyme-based techniques (see, e.g., section herein entitled“Expression in Alternate Systems”). For convenience, the phrase“post-translational modification,” when directed to the use of anaccelerant to form an oxime bond on a non-natural amino acidpolypeptide, includes non-natural amino acid polypeptides formed by anytechnique, including any in vivo and in vitro techniques, such as thosedescribed herein and known to those of ordinary skill in the art.

The ability to incorporate non-natural amino acids into recombinantproteins broadly expands the chemistries which may be implemented forderivatization. More specifically, protein derivatization to form anoxime bond on a non-natural amino acid portion of a polypeptide offersseveral advantages. First, the naturally occurring amino acids generallydo not form oxime bonds and thus reagents designed to form oxime bondswill react site-specifically with the non-natural amino acid componentof the polypeptide (assuming of course that the non-natural amino acidand the corresponding reagent have been designed to form an oxime bond),thus the ability to site-selectively derivatize proteins provides asingle homogeneous product as opposed to the mixtures of derivatizedproteins produced using prior art technology. Second, oxime adducts arestable under biological conditions, suggesting that proteins derivatizedby oxime exchange are valid candidates for therapeutic applications.Third, the stability of the resulting oxime bond can be manipulatedbased on the identity (i.e., the functional groups and/or structure) ofthe non-natural amino acid to which the oxime bond has been formed.Thus, in some embodiments, the oxime bond to the non-natural amino acidpolypeptide has a decomposition half life less than one hour, in otherembodiments less than 1 day, in other embodiments less than 2 days, inother embodiments less than 1 week and in other embodiments more than 1week. In yet other embodiments, the resulting oxime is stable for atleast two weeks under mildly acidic conditions, in other embodiments theresulting oxime is stable for at least 5 days under mildly acidicconditions. In other embodiments, the non-natural amino acid polypeptideis stable for at least 1 day in a pH between about 2 and 8; in otherembodiments, from a pH between about 2 to 6; in other embodiment, in apH between about 2 to 4. In other embodiments, using the strategies,methods, compositions and techniques described herein, one of ordinaryskill in the art will be able to synthesize an oxime bond to anon-natural amino acid polypeptide with a decomposition half-life tunedto the needs of that skilled artisan (e.g., for a therapeutic use suchas sustained release, or a diagnostic use, or an industrial use or amilitary use).

The formation of an oxime-containing non-natural amino acid ornon-natural amino acid polypeptide from the reaction of (a) acarbonyl-containing non-natural amino acid or carbonyl-containingnon-natural amino acid polypeptide and a hydroxylamine-containingreagent, or (b) a hydroxylamine-containing non-natural amino acid orhydroxylamine-containing non-natural amino acid polypeptide and acarbonyl-containing reagent, can be enhanced by addition of anaccelerant to the reaction mixture. An accelerant is a compound that hasat least one of the following properties: (a) increase the rate ofreaction between a carbonyl-containing compound and ahydroxylamine-containing compound to form an oxime-containing compound,where the increase in rate is relative to the reaction in the absence ofthe accelerant; (b) lower the activation energy of the reaction betweena carbonyl-containing compound and a hydroxylamine-containing compoundto form an oxime-containing compound, where the decrease in activationenergy is relative to the reaction in the absence of the accelerant; (c)increase the yield of an oxime-containing compound from the reaction ofa carbonyl-containing compound with a hydroxylamine-containing compound,where the increase in yield is relative to the reaction in the absenceof the accelerant; (d) lower the temperature at which acarbonyl-containing compound reacts with a hydroxylamine-containingcompound to form an oxime-containing compound, where the decrease intemperature is relative to the reaction in the absence of theaccelerant; (e) decrease the time necessary to react acarbonyl-containing compound with a hydroxylamine-containing compound toform an oxime-containing compound, wherein the decrease in time isrelative to the reaction in the absence of accelerant; (f) decrease theamount of reagents necessary to form an oxime group on a non-naturalamino acid polypeptide, wherein the decrease in amount of reagents isrelative to the reaction in the absence of accelerant; (g) decrease theside products resulting from the reaction of a carbonyl-containingcompound with a hydroxylamine-containing compound to form anoxime-containing compound, wherein the decrease in side products isrelative to the reaction in the absence of accelerant; (h) does notirreversibly destroy the tertiary structure of a polypeptide undergoingan oxime-forming reaction in the presence of an accelerant (excepting,of course, where the purpose of the reaction is to destroy such tertiarystructure); (i) can be separated from an oxime-containing compound invacuo; and (j) modulate the reaction of a carbonyl-containing compoundwith a hydroxylamine-containing compound. In further embodiments, theaccelerant has at least two of the aforementioned properties, three ofthe aforementioned properties, four of the aforementioned properties,five of the aforementioned properties, six of the aforementionedproperties, seven of the aforementioned properties, eight of theaforementioned properties, nine of the aforementioned properties, or allof the aforementioned properties. In a further embodiment, theaccelerant has none of the aforementioned properties.

The use of an accelerant includes the use of a single accelerant ormultiple accelerants. In addition, the molar ratio of accelerant tocarbonyl-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the molar ratio of accelerant tohydroxylamine-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the accelerant includes compounds that can besubstantially removed in vacuo from the resulting oxime-containingcompound. Further, the accelerant includes compounds containing adiamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazidemoiety.

Further, in any of the aforementioned aspects or embodiments, theaccelerant is selected from the group consisting of bifunctionalaromatic amines, oxoamine derivatives, and compounds having thefollowing structures:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), C(═NH)—NH and SO, SO₂.

In a further embodiment, the accelerant is a bifunctional aromaticamine. In a further embodiment, the aromatic amine is selected from thegroup:

Bifunctional Aromatic Amines:

In a further embodiment, the accelerant is an oxoamine derivative. In afurther embodiment, the oxoamine derivative is selected from the group:

Oxoamine Derivatives:

Further, the accelerant include compounds selected from the groupconsisting of:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), and C(—NH)—NH. Further, in any of theaforementioned aspects or embodiments, the accelerant is selected fromthe compounds presented in FIG. 5, FIG. 9, OT FIG. 10, including by wayof example any of compounds 6, 8, 10, 7, and 20 of FIG. 5. In any of theaforementioned aspects or embodiments, the accelerant includes an agentthat can form a hydrazone upon reaction with a carbonyl-containinggroup. Further, in any of the aforementioned aspects the accelerantactivity depends on the rate of reaction with the ketone moiety and thestability of the resulting intermediate. Further, in any of theaforementioned aspects or embodiments, the pH of the reaction mixturecomprising the accelerant, the carbonyl-containing compound and thehydroxylamine-containing compound is between about 2.0 and 10; betweenabout 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0;between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; betweenabout 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0;between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0and 6.5, between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0;about 5.5; about 6.0; about 6.5; and about 7.0.

The non-natural amino acid polypeptides described above are useful for,including but not limited to, novel therapeutics, diagnostics, catalyticenzymes, industrial enzymes, binding proteins (including but not limitedto, antibodies and antibody fragments), and including but not limitedto, the study of protein structure and function. See, e.g., Dougherty,(2000) Unnatural Amino Acids as Probes of Protein Structure andFunction, Current Opinion in Chemical Biology, 4:645-652. Other uses forthe non-natural amino acid polypeptides described above include, by wayof example only, assay-based, cosmetic, plant biology, environmental,energy-production, and/or military uses. However, the non-natural aminoacid polypeptides described above can undergo further modifications soas to incorporate new or modified functionalities, includingmanipulating therapeutic effectiveness of the polypeptide, improving thesafety profile of the polypeptide, adjusting the pharmacokinetics,pharmacologics and/or pharmacodynamics of the polypeptide (e.g.,increasing water solubility, bioavailability, increasing serumhalf-life, increasing therapeutic half-life, modulating immunogenicity,modulating biological activity, or extending the circulation time),providing additional functionality to the polypeptide, incorporating atag, label or detectable signal into the polypeptide, easing theisolation properties of the polypeptide, and any combination of theaforementioned modifications.

The methods, compositions, strategies and techniques described hereinare not limited to a particular type, class or family of polypeptides orproteins. Indeed, virtually any polypeptides may include at least onenon-natural amino acids described herein. By way of example only, thepolypeptide can be homologous to a therapeutic protein selected from thegroup consisting of: alpha-1 antitrypsin, angiostatin, antihemolyticfactor, antibody, antibody fragments, apolipoprotein, apoprotein, atrialnatriuretic factor, atrial natriuretic polypeptide, atrial peptide,C—X—C chemoline, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10,GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CCchemokine, monocyte chemoattractant protein-1, monocyte chemoattractantprotein-2, monocyte chemoattractant protein-3, monocyte inflammatoryprotein-1 alpha, monocyte inflammatory protein-1 beta, RANTES, 1309,R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kitligand, collagen, colony stimulating factor (CSF), complement factor 5a,complement inhibitor, complement receptor 1, cytokine, epithelialneutrophil activating peptide-78, MIP-16, MCP-1, epidermal growth factor(EGF), epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, anyinterferon-like molecule or member of the IFN family, interleukin (IL),IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,FLT, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone. The non-natural amino acid polypeptide may also behomologous to any polypeptide member of the growth hormone supergenefamily.

Such modifications include the incorporation of further functionalityonto the non-natural amino acid component of the polypeptide, includingbut not limited to, a desired functionality.

Thus, by way of example only, a non-natural amino acid polypeptidecontaining any one of the following amino acids may be further modifiedin the presence of the accelerants described herein using the methodsand compositions described herein:

(a)wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, lower        heteroalkylene, substituted lower heteroalkylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N—, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;

-   -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   each R″ is independently H, alkyl, substituted alkyl, or a        protecting group, or when more than one R″ group is present, two        R″ optionally form a heterocycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin;    -   each of R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   or the -A-B-J-R groups together form a bicyclic or tricyclic        cycloalkyl or heterocycloalkyl comprising at least one carbonyl        group, including a dicarbonyl group, protected carbonyl group,        including a protected dicarbonyl group, or masked carbonyl        group, including a masked dicarbonyl group;    -   or the -J-R group together forms a monocyclic or bicyclic        cycloalkyl or heterocycloalkyl comprising at least one carbonyl        group, including a dicarbonyl group, protected carbonyl group,        including a protected dicarbonyl group, or masked carbonyl        group, including a masked dicarbonyl group;

(b)wherein:

-   -   R is alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin; and    -   each R_(a) is independently selected from the group consisting        of H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′        where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where        each R′ is independently H, alkyl, or substituted alkyl.

(c)wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, lower        heteroalkylene, substituted lower heteroalkylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;    -   K is —NR₆R₇ or —N═CR₆R₇;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin;    -   each of R₃ and R_(a) is independently H, halogen, lower alkyl,        or substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   each of R₆ and R₇ is independently selected from the group        consisting of H, alkyl, substituted alkyl, alkenyl, substituted        alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide,        substituted polyalkylene oxide, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, alkaryl, substituted        alkaryl, aralkyl, and substituted aralkyl, —C(O)R″, —C(O)₂R″,        —C(O)N(R″)₂, wherein each R″ is independently hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkoxy,        substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,        substituted alkaryl, aralkyl, or substituted aralkyl; or R₆ or        R₇ is L-X, where X is a selected from the group consisting of a        desired functionality; and L is optional, and when present is a        linker selected from the group consisting of alkylene,        substituted alkylene, alkenylene, substituted alkenylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;

(d)

-   -   wherein;    -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin, amino acid, polypeptide, or polynucleotide;    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin, amino acid, polypeptide, or polynucleotide;

X₁ is C, S, or S(O); and L is alkylene, substituted arylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where each R′ isindependently H, alkyl, or substituted alkyl; or

(e)wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;

-   -    indicates bonding to the A group and (b) indicates bonding to        respective carbonyl groups, R₃ and R₄ are independently chosen        from H, halogen, alkyl, substituted alkyl, cycloalkyl, or        substituted cycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄        groups optionally form a cycloalkyl or a heterocycloalkyl;    -   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or        substituted cycloalkyl;    -   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,        substituted alkyl, cycloalkyl, or substituted cycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin, amino acid, polypeptide, or polynucleotide; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin, amino acid, polypeptide, or polynucleotide.

In one aspect of the methods and compositions described herein arecompositions that include at least one protein with at least one,including but not limited to, at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten or more non-natural amino acids that havebeen post-translationally modified. The post-translationally-modifiednon-natural amino acids can be the same or different, including but notlimited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more different sites in the protein thatcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more different post-translationally-modified non-naturalamino acids. In another aspect, a composition includes a protein with atleast one, but fewer than all, of a particular amino acid present in theprotein is substituted with the post-translationally-modifiednon-natural amino acid. For a given protein with more than onepost-translationally-modified non-natural amino acids, thepost-translationally-modified non-natural amino acids can be identicalor different (including but not limited to, the protein can include twoor more different types of post-translationally-modified non-naturalamino acids, or can include two of the samepost-translationally-modified non-natural amino acid). For a givenprotein with more than two post-translationally-modified non-naturalamino acids, the post-translationally-modified non-natural amino acidscan be the same, different or a combination of a multiplepost-translationally-modified non-natural amino acid of the same kindwith at least one different post-translationally-modified non-naturalamino acid.

A. Methods for Post-Translationally Modifying Non-Natural Amino AcidPolypeptides in the Presence of at Least One Accelerant: Reactions ofCarbonyl-Containing Non-Natural Amino Acids withHydroxylamine-Containing Reagents

The sidechains of the naturally occurring amino acids lack highlyelectrophilic sites. Therefore, the incorporation of an non-naturalamino acid with an electrophile-containing sidechain, including, by wayof example only, an amino acid containing a carbonyl or dicarbonyl groupsuch as a ketone, makes possible the site-specific derivatization ofthis sidechain via nucleophilic attack of the carbonyl or dicarbonylgroup. In the instance where the attacking nucleophile is ahydroxylamine, an oxime-derivatized protein will be generated. Themethods for derivatizing and/or further modifying may be conducted witha polypeptide that has been purified prior to the derivatization step orafter the derivatization step. Further, the derivatization step canoccur under mildly acidic to slightly basic conditions, including by wayof example, between a pH of about 2-8, or between a pH of about 4-8.

The formation of an oxime-containing non-natural amino acid ornon-natural amino acid polypeptide from the reaction of acarbonyl-containing non-natural amino acid or carbonyl-containingnon-natural amino acid polypeptide and a hydroxylamine-containingreagent can be enhanced by addition of an accelerant to the reactionmixture. An accelerant is a compound that has at least one of thefollowing properties: (a) increase the rate of reaction between acarbonyl-containing compound and a hydroxylamine-containing compound toform an oxime-containing compound, where the increase in rate isrelative to the reaction in the absence of the accelerant; (b) lower theactivation energy of the reaction between a carbonyl-containing compoundand a hydroxylamine-containing compound to form an oxime-containingcompound, where the decrease in activation energy is relative to thereaction in the absence of the accelerant; (c) increase the yield of anoxime-containing compound from the reaction of a carbonyl-containingcompound with a hydroxylamine-containing compound, where the increase inyield is relative to the reaction in the absence of the accelerant; (d)lower the temperature at which a carbonyl-containing compound reactswith a hydroxylamine-containing compound to form an oxime-containingcompound, where the decrease in temperature is relative to the reactionin the absence of the accelerant; (e) decrease the time necessary toreact a carbonyl-containing compound with a hydroxylamine-containingcompound to form an oxime-containing compound, wherein the decrease intime is relative to the reaction in the absence of accelerant; (f)decrease the amount of reagents necessary to form an oxime group on anon-natural amino acid polypeptide, wherein the decrease in amount ofreagents is relative to the reaction in the absence of accelerant; (g)decrease the side products resulting from the reaction of acarbonyl-containing compound with a hydroxylamine-containing compound toform an oxime-containing compound, wherein the decrease in side productsis relative to the reaction in the absence of accelerant; (h) does notirreversibly destroy the tertiary structure of a polypeptide undergoingan oxime-forming reaction in the presence of an accelerant (excepting,of course, where the purpose of the reaction is to destroy such tertiarystructure); (i) can be separated from an oxime-containing compound invacuo; and (j) modulate the reaction of a carbonyl-containing compoundwith a hydroxylamine-containing compound. In further embodiments, theaccelerant has at least two of the aforementioned properties, three ofthe aforementioned properties, four of the aforementioned properties,five of the aforementioned properties, six of the aforementionedproperties, seven of the aforementioned properties, eight of theaforementioned properties, nine of the aforementioned properties, or allof the aforementioned properties. In a further embodiment, theaccelerant has none of the aforementioned properties.

The use of an accelerant includes the use of a single accelerant ormultiple accelerants. In addition, the molar ratio of accelerant tocarbonyl-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the molar ratio of accelerant tohydroxylamine-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the accelerant includes compounds that can besubstantially removed in vacuo from the resulting oxime-containingcompound. Further, the accelerant includes compounds containing adiamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazidemoiety.

Further, in any of the aforementioned aspects or embodiments, theaccelerant is selected from the group consisting of bifunctionalaromatic amines, oxoamine derivatives, and compounds having thefollowing structures:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(—NH), C(═NH)—NH and SO, SO₂.

In a further embodiment, the accelerant is a bifunctional aromaticamine. In a further embodiment, the aromatic amine is selected from thegroup:

Bifunctional Aromatic Amines:

In a further embodiment, the accelerant is an oxoamine derivative. In afurther embodiment, the oxoamine derivative is selected from the group:

Oxoamine Derivatives:

Further, the accelerant include compounds selected from the groupconsisting of:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), and C(═NH)—NH. Further, in any of theaforementioned aspects or embodiments, the accelerant is selected fromthe compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by wayof example any of compounds 6, 8, 10, 7, and 20 of FIG. 5. In any of theaforementioned aspects or embodiments, the accelerant includes an agentthat can form a hydrazone upon reaction with a carbonyl-containinggroup. Further, in any of the aforementioned aspects the accelerantactivity depends on the rate of reaction with the ketone moiety and thestability of the resulting intermediate. Further, in any of theaforementioned aspects or embodiments, the pH of the reaction mixturecomprising the accelerant, the carbonyl-containing compound and thehydroxylamine-containing compound is between about 2.0 and 10; betweenabout 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0;between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; betweenabout 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0;between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0;about 5.5; about 6.0; about 6.5; and about 7.0.

A protein-derivatizing method based upon the reaction of a carbonyl- ordicarbonyl-containing protein with a hydroxylamine-substituted moleculehas distinct advantages. First, hydroxylamines undergo condensation withcarbonyl- or dicarbonyl-containing compounds in a pH between about 2 and8 (and in further embodiments in a pH between about 4 and 8) to generateoxime adducts. Under these conditions, the sidechains of the naturallyoccurring amino acids are unreactive. Second, such selective chemistrymakes possible the site-specific derivatization of recombinant proteins:derivatized proteins can now be prepared as defined homogeneousproducts. Third, the mild conditions needed to effect the reaction ofthe hydroxylamines described herein with the carbonyl- ordicarbonyl-containing polypeptides described herein generally do notirreversibly destroy the tertiary structure of the polypeptide(excepting, of course, where the purpose of the reaction is to destroysuch tertiary structure). Finally, although the hydroxylamine groupamino appears to be metabolized by E. coli, the condensation ofhydroxylamines with carbonyl- or dicarbonyl-containing moleculesgenerates oxime adducts which are stable under biological conditions.

By way of example only, the following non-natural amino acids are thetype of carbonyl- or dicarbonyl-containing amino acids that are reactivewith the hydroxylamine-containing reagents described herein to form anoxime-containing non-natural amino acid or polypeptide in the presenceof an accelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, lower        heteroalkylene, substituted lower heteroalkylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;

-   -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   each R″ is independently H, alkyl, substituted alkyl, or a        protecting group, or when more than one R″ group is present, two        R″ optionally form a heterocycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin;    -   each of R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   or the -A-B-J-R groups together form a bicyclic or tricyclic        cycloalkyl or heterocycloalkyl comprising at least one carbonyl        group, including a dicarbonyl group, protected carbonyl group,        including a protected dicarbonyl group, or masked carbonyl        group, including a masked dicarbonyl group;    -   or the -J-R group together forms a monocyclic or bicyclic        cycloalkyl or heterocycloalkyl comprising at least one carbonyl        group, including a dicarbonyl group, protected carbonyl group,        including a protected dicarbonyl group, or masked carbonyl        group, including a masked dicarbonyl group.

By way of example only, for the aforementioned purposes, compounds ofFormula (I) include compounds

having the structure:wherein:

-   -   R is alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin; and    -   each R_(a) is independently selected from the group consisting        of H, halogen, alkyl, substituted alkyl N(R′)₂, C(O)_(k)R′ where        k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each        R′ is independently H, alkyl, or substituted alkyl.

By way of example only, for the aforementioned purposes, compounds ofFormula (I) include compounds

having the structure:

-   -   wherein;    -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   R is alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin, amino acid, polypeptide, or polynucleotide;    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin, amino acid, polypeptide, or polynucleotide;    -   X₁ is C, S, or S(O); and L is a bond, alkylene, substituted        alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where        each R′ is independently H, alkyl, or substituted alkyl.

By way of further example only, for the aforementioned purposes,compounds of Formula (I) include compounds having the structure ofFormula (XXXX):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;

-   -    indicates bonding to the A group and (b) indicates bonding to        respective carbonyl groups, R₃ and R₄ are independently chosen        from H, halogen, alkyl, substituted alkyl, cycloalkyl, or        substituted cycloalkyl, or R₃ and R₄ or two R₃ groups or two R        groups optionally form a cycloalkyl or a heterocycloalkyl;    -   R is alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,        substituted alkyl, cycloalkyl, or substituted cycloalkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin, amino acid, polypeptide, or polynucleotide; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin, amino acid, polypeptide, or polynucleotide.

The types of polypeptides that comprise such carbonyl- ordicarbonyl-containing non-natural amino acids is practically unlimitedas long as the carbonyl- or dicarbonyl-containing non-natural amino acidis located on the polypeptide so that the hydroxylamine reagent canreact with the carbonyl or dicarbonyl group and not create a resultingmodified non-natural amino acid that destroys the tertiary structure ofthe polypeptide (excepting, of course, if such destruction is thepurpose of the reaction).

By way of example only, the following hydroxylamine-containing reagentsare the type of hydroxylamine-containing reagents that are reactive withthe carbonyl- or dicarbonyl-containing non-natural amino acids describedherein to form an oxime-containing non-natural amino acid in thepresence of an accelerant described herein (although such a reaction maybe less efficient in the absence of an accelerant described herein):

wherein:

-   -   each X is independently H, alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,        substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,        polyalkylene oxide, substituted polyalkylene oxide, aryl,        substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,        substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or        substituted alkylene)-ON(R″)₂, -(alkylene or substituted        alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl        or substituted aryl), —C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein        each R″ is independently hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,        substituted aryl, heteroaryl, alkaryl, substituted alkaryl,        aralkyl, or substituted aralkyl;    -   or each X is independently selected from the group consisting of        a desired functionality;    -   each L is independently selected from the group consisting of        alkylene, substituted alkylene, alkenylene, substituted        alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,        —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1,        2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, -(alkylene or        substituted alkylene)NR′C(O)O-(alkylene or substituted        alkylene)-, —O—CON(R′)-(alkylene or substituted alkylene)-,        —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,        —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,        —N(R′)C(O)O-(alkylene or substituted alkylene)-,        —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or        substituted alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—;    -   L₁ is optional, and when present, is        —C(R′)_(p)—NR′—C(O)O-(alkylene or substituted alkylene)- where p        is 0, 1, or 2,    -   each R′ is independently H, alkyl, or substituted alkyl;    -   W is —N(R₈)₂, where each R₈ is independently H or an amino        protecting group; and n is 1 to 3; provided that L-L₁-W together        provide at least one hydroxylamine group capable of reacting        with a carbonyl (including a dicarbonyl) group on a non-natural        amino acid or a (modified) non-natural amino, acid polypeptide.

In an illustrative embodiment, a hydroxylamine-derivatized reagent isadded to a buffered solution (pH 2-8) of a carbonyl-containingnon-natural amino acid polypeptide and an accelerant. The resultingoxime-containing non-natural amino acid polypeptide is purified by HPLC,FPLC or size-exclusion chromatography.

In a further or alternative illustrative embodiment, the molar ratio ofa compound of Formula (I) to a compound of Formula (XIX) is about 1:2;1:1; 1.5:1; 1.5:2; 2:1; 1:1.5; 2:1.5; or 1.5 to 2.

In one embodiment, multiple linker chemistries can reactsite-specifically with a carbonyl- or dicarbonyl-substituted non-naturalamino acid polypeptide to form an oxime bond in the presence of anaccelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). In oneembodiment, the linker methods described herein utilize linkerscontaining the hydroxylamine functionality on at least one linkertermini (mono, bi- or multi-functional). The condensation of ahydroxylamine-derivatized linker with a keto-substituted proteingenerates a stable oxime bond. Bi- and/or multi-functional linkers(e.g., hydroxylamine with one, or more, other linking chemistries) allowthe site-specific connection of different molecules (e.g., otherproteins, polymers or small molecules) to the non-natural amino acidpolypeptide, while mono-functional linkers (hydroxylamine-substituted onall termini) facilitate the site-specific dimer- or oligomerization ofthe non-natural amino acid polypeptide. By combining this linkerstrategy with the in vivo translation technology described herein, itbecomes possible to specify the three-dimensional structures ofchemically-elaborated proteins.

B. Methods for Post-Translationally Modifying Non-Natural Amino AcidPolypeptides in the Presence of at Least One Accelerant: Reactions ofHydroxylamine-Containing Non-Natural Amino Acids withCarbonyl-Containing Reagents

The post-translational modification techniques and compositionsdescribed above may also be used with hydroxylamine-containingnon-natural amino acids reacting with carbonyl- or dicarbonyl-containingreagents to produce modified oxime-containing non-natural amino acidpolypeptides in the presence of an accelerant described herein (althoughsuch a reaction may be less efficient in the absence of an accelerantdescribed herein).

The formation of an oxime-containing non-natural amino acid ornon-natural amino acid polypeptide from the reaction of ahydroxylamine-containing non-natural amino acid orhydroxylamine-containing non-natural amino acid polypeptide and acarbonyl-containing reagent can be enhanced by addition of an accelerantto the reaction mixture. An accelerant is a compound that has at leastone of the following properties: (a) increase the rate of reactionbetween a carbonyl-containing compound and a hydroxylamine-containingcompound to form an oxime-containing compound, where the increase inrate is relative to the reaction in the absence of the accelerant; (b)lower the activation energy of the reaction between acarbonyl-containing compound and a hydroxylamine-containing compound toform an oxime-containing compound, where the decrease in activationenergy is relative to the reaction in the absence of the accelerant; (c)increase the yield of an oxime-containing compound from the reaction ofa carbonyl-containing compound with a hydroxylamine-containing compound,where the increase in yield is relative to the reaction in the absenceof the accelerant; (d) lower the temperature at which acarbonyl-containing compound reacts with a hydroxylamine-containingcompound to form an oxime-containing compound, where the decrease intemperature is relative to the reaction in the absence of theaccelerant; (e) decrease the time necessary to react acarbonyl-containing compound with a hydroxylamine-containing compound toform an oxime-containing compound, wherein the decrease in time isrelative to the reaction in the absence of accelerant; (f) decrease theamount of reagents necessary to form an oxime group on a non-naturalamino acid polypeptide, wherein the decrease in amount of reagents isrelative to the reaction in the absence of accelerant; (g) decrease theside products resulting from the reaction of a carbonyl-containingcompound with a hydroxylamine-containing compound to form anoxime-containing compound, wherein the decrease in side products isrelative to the reaction in the absence of accelerant; (h) does notirreversibly destroy the tertiary structure of a polypeptide undergoingan oxime-forming reaction in the presence of an accelerant (excepting,of course, where the purpose of the reaction is to destroy such tertiarystructure); (i) can be separated from an oxime-containing compound invacuo; and (j) modulate the reaction of a carbonyl-containing compoundwith a hydroxylamine-containing compound. In further embodiments, theaccelerant has at least two of the aforementioned properties, three ofthe aforementioned properties, four of the aforementioned properties,five of the aforementioned properties, six of the aforementionedproperties, seven of the aforementioned properties, eight of theaforementioned properties, nine of the aforementioned properties, or allof the aforementioned properties. In a further embodiment, theaccelerant has none of the aforementioned properties.

The use of an accelerant includes the use of a single accelerant ormultiple accelerants. In addition, the molar ratio of accelerant tocarbonyl-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the molar ratio of accelerant tohydroxylamine-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the accelerant includes compounds that can besubstantially removed in vacuo from the resulting oxime-containingcompound. Further, the accelerant includes compounds containing adiamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazidemoiety.

Further, in any of the aforementioned aspects or embodiments, theaccelerant is selected from the group consisting of bifunctionalaromatic amines, oxoamine derivatives, and compounds having thefollowing structures:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-aryl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), C(═NH)—NH and SO, SO₂.

In a further embodiment, the accelerant is a bifunctional aromaticamine. In a further embodiment, the aromatic amine is selected from thegroup:

Bifunctional Aromatic Amines:

In a further embodiment, the accelerant is an oxoamine derivative. In afurther embodiment, the oxoamine derivative is selected from the group:

Oxoamine Derivatives:

Further, the accelerant include compounds selected from the groupconsisting of:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L, is a bond,C(═O), C(═NH), and C(═NH)—NH. Further, in any of the aforementionedaspects or embodiments, the accelerant is selected from the compoundspresented in FIG. 5, FIG. 9, or FIG. 10, including by way of example anyof compounds 6, 8, 10, 7, and 20 of FIG. 5. In any of the aforementionedaspects or embodiments, the accelerant includes an agent that can form ahydrazone upon reaction with a carbonyl-containing group. Further, inany of the aforementioned aspects the accelerant activity depends on therate of reaction with the ketone moiety and the stability of theresulting intermediate. Further, in any of the aforementioned aspects orembodiments, the pH of the reaction mixture comprising the accelerant,the carbonyl-containing compound and the hydroxylamine-containingcompound is between about 2.0 and 10; between about 2.0 and 9.0; betweenabout 2.0 and 8.0; between about 3.0 and 7.0; between about 4.0 and 6.0;between about 3.0 and 10.0; between about 4.0 and 10.0; between about3.0 and 9.0; between about 3.0 and 8.0; between about 2.0 and 7.0;between about 3.0 and 6.0; between about 4.0 and 9.0; between about 4.0and 8.0; between about 4.0 and 7.0; between about 4.0 and 6.5; betweenabout 4.5 and 6.5; about 4.0; about 4.5; about 5.0; about 5.5; about6.0; about 6.5; and about 7.0.

A protein-derivatizing method based upon the reaction of ahydroxylamine-containing protein with a carbonyl- ordicarbonyl-substituted molecule has distinct advantages. First,hydroxylamines undergo condensation with carbonyl- ordicarbonyl-containing compounds in a pH between about 2 to 8 (and infurther embodiments in a pH between about 4 to 8) to generate oximeadducts. Under these conditions, the sidechains of the naturallyoccurring amino acids are unreactive. Second, such selective chemistrymakes possible the site-specific derivatization of recombinant proteins:derivatized proteins can now be prepared as defined homogeneousproducts. Third, the mild conditions needed to effect the reaction ofthe carbonyl- or dicarbonyl-containing reagents described herein withthe hydroxylamine-containing polypeptides described herein generally donot irreversibly destroy the tertiary structure of the polypeptide(excepting, of course, where the purpose of the reaction is to destroysuch tertiary structure). Finally, although the hydroxylamine groupamino appears to be metabolized by E. coli, the condensation ofcarbonyl- or dicarbonyl-containing reagents withhydroxylamine-containing amino acids generates oxime adducts which arestable under biological conditions.

By way of example only, the following non-natural amino acids are thetype of hydroxylamine-containing amino acids that are reactive with thecarbonyl- or dicarbonyl-containing reagents described herein to form anoxime-containing non-natural amino acid or polypeptide in the presenceof an accelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, lower        heteroalkylene, substituted lower heteroalkylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;    -   K is —NH₂;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin, amino acid, polypeptide, or polynucleotide; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin, amino acid, polypeptide, or polynucleotide;    -   each of R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl.

The types of polypeptides that comprise such hydroxylamine-containingnon-natural amino acids is practically unlimited as long as thehydroxylamine-containing non-natural amino acid is located on thepolypeptide so that the carbonyl- or dicarbonyl-containing reagent canreact with the hydroxylamine group and not create a resulting modifiednon-natural amino acid that destroys the tertiary structure of thepolypeptide (excepting, of course, if such destruction is the purpose ofthe reaction).

By way of example only, the following carbonyl- or dicarbonyl-containingreagents are the type of carbonyl- or dicarbonyl-containing reagentsthat are reactive with the hydroxylamine-containing non-natural aminoacids described herein to form an oxime-containing non-natural aminoacid or polypeptide in the presence of an accelerant described herein(although such a reaction may be less efficient in the absence of anaccelerant described herein):

wherein:

-   -   each X is independently H, alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,        substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,        polyalkylene oxide, substituted polyalkylene oxide, aryl,        substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,        substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or        substituted alkylene)-ON(R″)₂, -(alkylene or substituted        alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl        or substituted aryl), —C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein        each R″ is independently hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,        substituted aryl, heteroaryl, alkaryl, substituted alkaryl,        aralkyl, or substituted aralkyl; or each X is independently        selected from the group consisting of a desired functionality;    -   each L is independently selected from the group consisting of        alkylene, substituted alkylene, alkenylene, substituted        alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,        —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1,        2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, -(alkylene or        substituted alkylene)NR′C(O)O-(alkylene or substituted        alkylene)-, —O—CON(R′)-(alkylene or substituted alkylene)-,        —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,        —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,        —N(R′)C(O)O-(alkylene or substituted alkylene)-,        —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or        substituted alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—;    -   L₁ is optional, and when present, is        —C(R′)_(p)—NR′—C(O)O-(alkylene or substituted alkylene)- where p        is 0, 1, or 2;    -   each R′ is independently H, alkyl, or substituted alkyl;    -   W is -J-R, where

-   -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl; each R″ is independently H, alkyl, substituted        alkyl, or a protecting group, or when more than one R″ group is        present, two R″ optionally form a heterocycloalkyl; and n is 1        to 3;    -   provided that L-L₁-W together provide at least one carbonyl        group (including a dicarbonyl group) capable of reacting with an        hydroxylamine group on a non-natural amino acid or a (modified)        non-natural amino acid polypeptide.

In an illustrative embodiment, a carbonyl-derivatized reagent is addedto a buffered solution (pH 2-8) of a hydroxylamine-containingnon-natural amino acid polypeptide and an accelerant. The resultingoxime-containing non-natural amino acid polypeptide is purified by HPLC,FPLC or size-exclusion chromatography.

In a further or alternative illustrative embodiment, the molar ratio ofa compound of Formula (XI) to a compound of Formula (XIX) is about 1:2;1:1; 1.5:1; 1.5:2; 2:1; 1:1.5; 2:1.5; or 1.5 to 2.

In one embodiment, multiple linker chemistries can reactsite-specifically with a hydroxylamine-substituted non-natural aminoacid polypeptide. In one embodiment, the linker methods described hereinutilize linkers containing the carbonyl or dicarbonyl functionality onat least one linker termini (mono, bi- or multi-functional). Thecondensation of a carbonyl- or dicarbonyl-derivatized linker with ahydroxylamine-substituted protein generates a stable oxime bond. Bi-and/or multi-functional linkers (e.g., carbonyl or dicarbonyl with one,or more, other linking chemistries) allow the site-specific connectionof different molecules (e.g., other proteins, polymers or smallmolecules) to the non-natural amino acid polypeptide, whilemono-functional linkers (carbonyl- or dicarbonyl-substituted on alltermini) facilitate the site-specific dimer- or oligomerization of thenon-natural amino acid polypeptide. By combining this linker strategywith the in vivo translation technology described herein, it becomespossible to specify the three-dimensional structures ofchemically-elaborated proteins.

C. Example of Adding Functionality in the Presence of at Least OneAccelerant: Macromolecular Polymers Coupled to Non-Natural Amino AcidPolypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide via an oxime bond formed in thepresence of an accelerant described herein (although such a reaction maybe less efficient in the absence of an accelerant described herein),including but not limited to, a desired functionality. As anillustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of ordinaryskill in the art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

The formation of a macromolecular polymer coupled via an oxime bond to anon-natural amino acid polypeptide from (a) the reaction of ahydroxylamine-containing non-natural amino acid polypeptide and acarbonyl-containing reagent, or (b) the reaction of acarbonyl-containing non-natural amino acid polypeptide and ahydroxylamine-containing reagent, can be enhanced by addition of anaccelerant to the reaction mixture. Such an accelerant is a compoundthat has at least one of the following properties: (a) increase the rateof reaction between a carbonyl-containing compound and ahydroxylamine-containing compound to form an oxime-containing compound,where the increase in rate is relative to the reaction in the absence ofthe accelerant; (b) lower the activation energy of the reaction betweena carbonyl-containing compound and a hydroxylamine-containing compoundto form an oxime-containing compound, where the decrease in activationenergy is relative to the reaction in the absence of the accelerant; (c)increase the yield of an oxime-containing compound from the reaction ofa carbonyl-containing compound with a hydroxylamine-containing compound,where the increase in yield is relative to the reaction in the absenceof the accelerant; (d) lower the temperature at which acarbonyl-containing compound reacts with a hydroxylamine-containingcompound to form an oxime-containing compound, where the decrease intemperature is relative to the reaction in the absence of theaccelerant; (e) decrease the time necessary to react acarbonyl-containing compound with a hydroxylamine-containing compound toform an oxime-containing compound, wherein the decrease in time isrelative to the reaction in the absence of accelerant; (f) decrease theamount of reagents necessary to form an oxime group on a non-naturalamino acid polypeptide, wherein the decrease in amount of reagents isrelative to the reaction in the absence of accelerant; (g) decrease theside products resulting from the reaction of a carbonyl-containingcompound with a hydroxylamine-containing compound to form anoxime-containing compound, wherein the decrease in side products isrelative to the reaction in the absence of accelerant; (h) does notirreversibly destroy the tertiary structure of a polypeptide undergoingan oxime-forming reaction in the presence of an accelerant (excepting,of course, where the purpose of the reaction is to destroy such tertiarystructure); (i) can be separated from an oxime-containing compound invacuo; and (j) modulate the reaction of a carbonyl-containing compoundwith a hydroxylamine-containing compound. In further embodiments, theaccelerant has at least two of the aforementioned properties, three ofthe aforementioned properties, four of the aforementioned properties,five of the aforementioned properties, six of the aforementionedproperties, seven of the aforementioned properties, eight of theaforementioned properties, nine of the aforementioned properties, or allof the aforementioned properties. In a further embodiment, theaccelerant has none of the aforementioned properties.

The use of an accelerant includes the use of a single accelerant ormultiple accelerants. In addition, the molar ratio of accelerant tocarbonyl-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the molar ratio of accelerant tohydroxylamine-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the accelerant includes compounds that can besubstantially removed in vacuo from the resulting oxime-containingcompound. Further, the accelerant includes compounds containing adiamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazidemoiety.

Further, in any of the aforementioned aspects or embodiments, theaccelerant is selected from the group consisting of bifunctionalaromatic amines, oxoamine derivatives, and compounds having thefollowing structures:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), C(═NH)—NH and SO, SO₂.

In a further embodiment, the accelerant is a bifunctional aromaticamine. In a further embodiment, the aromatic amine is selected from thegroup:

Bifunctional Aromatic Amines:

In a further embodiment, the accelerant is an oxoamine derivative. In afurther embodiment, the oxoamine derivative is selected from the group:

Oxoamine Derivatives:

Further, the accelerant include compounds selected from the groupconsisting of:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), and C(═NH)—NH. Further, in any of theaforementioned aspects or embodiments, the accelerant is selected fromthe compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by wayof example any of compounds 6, 8, 10, 7, and 20 of FIG. 5. In any of theaforementioned aspects or embodiments, the accelerant includes an agentthat can form a hydrazone upon reaction with a carbonyl-containinggroup. Further, in any of the aforementioned aspects the accelerantactivity depends on the rate of reaction with the ketone moiety and thestability of the resulting intermediate. Further, in any of theaforementioned aspects or embodiments, the pH of the reaction mixturecomprising the accelerant, the carbonyl-containing compound and thehydroxylamine-containing compound is between about 2.0 and 10; betweenabout 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0;between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; betweenabout 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0;between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0;about 5.5; about 6.0; about 6.5; and about 7.0.

A wide variety of macromolecular polymers and other molecules can becoupled to the non-natural amino acid polypeptides described herein tomodulate biological properties of the non-natural amino acid polypeptide(or the corresponding natural amino acid polypeptide), and/or providenew biological properties to the non-natural amino acid polypeptide (orthe corresponding natural amino acid polypeptide). These macromolecularpolymers can be coupled to the non-natural amino acid polypeptide via anoxime bond on the non-natural amino acid.

Water soluble polymers can be coupled to the non-natural amino acidpolypeptides described herein. The water soluble polymer may be coupledto the non-natural amino acid by an oxime bond. In some cases, thenon-natural amino acid polypeptides described herein comprise one ormore non-natural amino acid(s) linked to water soluble polymers and oneor more naturally-occurring amino acids linked to water solublepolymers. Covalent attachment of hydrophilic polymers to a biologicallyactive molecule represents one approach to increasing water solubility(such as in a physiological environment), bioavailability, increasingserum half-life, increasing therapeutic half-life, modulatingimmunogenicity, modulating biological activity, or extending thecirculation time of the biologically active molecule, includingproteins, peptides, and particularly hydrophobic molecules. Additionalimportant features of such hydrophilic polymers includebiocompatibility, lack of toxicity, and lack of immunogenicity.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

Examples of suitable hydrophilic polymers include: polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethylene glycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinylalcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing. The water soluble polymer maybe any structural form including but not limited to linear, forked orbranched. In some embodiments, polymer backbones that are water-soluble,with from 2 to about 300 termini, are particularly useful.Multifunctional polymer derivatives include, but are not limited to,linear polymers having two termini, each terminus being bonded to afunctional group which may be the same or different. In someembodiments, the water polymer comprises a poly(ethylene glycol) moiety.The molecular weight of the polymer may be of a wide range, includingbut not limited to, between about 100 Da and about 100,000 Da or more.The molecular weight of the polymer may be between about 100 Da andabout 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da,800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. Insome embodiments, the molecular weight of the polymer is between about100 Da and about 50,000 Da. In some embodiments, the molecular weight ofthe polymer is between about 100 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 1,000Da and about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and about 40,000 Da. In some embodiments, the poly(ethylene glycol)molecule is a branched polymer. The molecular weight of the branchedchain PEG may be between about 1,000 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 1,000 Da and about50,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 1,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the branched chain PEG is betweenabout 5,000 Da and about 40,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 5,000 Da and about20,000 Da. Those of ordinary skill in the art will recognize that theforegoing list for substantially water soluble backbones is by no meansexhaustive and is merely illustrative, and that all polymeric materialshaving the qualities described above are contemplated as being suitablefor use in methods and compositions described herein.

As described above, one example of a hydrophilic polymer ispoly(ethylene glycol), abbreviated PEG, which has been used extensivelyin pharmaceuticals, on artificial implants, and in other applicationswhere biocompatibility, lack of toxicity, and lack of immunogenicity areof importance. The polymer:polyeptide embodiments described herein willuse PEG as an example hydrophilic polymer with the understanding thatother hydrophilic polymers may be similarly utilized in suchembodiments.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). PEGis typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects.

The term “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented as linked to a non-natural amino acidpolypeptide by the formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group. The term PEG includes, but is not limited to,poly(ethylene glycol) in any of its forms, including bifunctional PEG,multiarmed PEG, derivatized PEG, forked PEG, branched PEG (with eachchain having a molecular weight of from about 1 kDa to about 100 kDa,from about 1 kDa to about 50 kDa, or from about 1 kDa to about 20 kDa),pendent PEG (i.e. PEG or related polymers having one or more functionalgroups pendent to the polymer backbone), or PEG with degradable linkagestherein. In one embodiment, PEG in which n is from about 20 to about2000 is suitable for use in the methods and compositions describedherein. In some embodiments, the water polymer comprises a poly(ethyleneglycol) moiety. The molecular weight of the polymer may be of a widerange, including but not limited to, between about 100 Da and about100,000 Da or more. The molecular weight of the polymer may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of the polymeris between about 100 Da and about 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and about 40,000Da. In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and about 40,000 Da. In some embodiments, the molecularweight of the polymer is between about 5,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of the polymer is between about10,000 Da and about 40,000 Da. In some embodiments, the poly(ethyleneglycol) molecule is a branched polymer. The molecular weight of thebranched chain PEG may be between about 1,000 Da and about 100,000 Da,including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da,50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 1,000 Da andabout 50,000 Da. In some embodiments, the molecular weight of thebranched chain PEG is between about 1,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 5,000 Da and about 40,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 5,000 Da andabout 20,000 Da. A wide range of PEG molecules are described in,including but not limited to, the Shearwater Polymers, Inc. catalog,Nektar Therapeutics catalog, incorporated herein by reference.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Malcromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Malcromol. Chem. 180:1381 (1979), succinimidyl ester(see, e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see,e.g., U.S. Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al.Eur. J. Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem.13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343(1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In some cases, a PEG terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). Alternatively, the PEG canterminate with a reactive group, thereby forming a bifunctional polymer.Typical reactive groups can include those reactive groups that arecommonly used to react with the functional groups found in the 20 commonamino acids (including but not limited to, maleimide groups, activatedcarbonates (including but not limited to, p-nitrophenyl ester),activated esters (including but not limited to, N-hydroxysuccinimide,p-nitrophenyl ester) and aldehydes) as well as functional groups thatare inert to the 20 common amino acids but that react specifically withcomplementary functional groups present in non-natural amino acids toform an oxime group in the presence of an accelerant described herein(although such a reaction may be less efficient in the absence of anaccelerant described herein); examples of the latter include but are notlimited to, carbonyl or dicarbonyl and hydroxylamine groups.

It is noted that the other end of the PEG, which is shown in the aboveformula by Y, will attach either directly or indirectly to a polypeptidevia a non-natural amino acid. When Y is a hydroxylamine group, then thehydroxylamine-containing PEG reagent can react with a carbonyl- ordicarbonyl-containing non-natural amino acid in a polypeptide to form aPEG group coupled to the polypeptide via an oxime bond in the presenceof an accelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein). When Y is acarbonyl or dicarbonyl group, then the carbonyl- ordicarbonyl-containing PEG reagent can react with ahydroxylamine-containing non-natural amino acid in a polypeptide to forma PEG group coupled to the polypeptide via an oxime bond in the presenceof an accelerant described herein (although such a reaction may be lessefficient in the absence of an accelerant described herein).

Heterobifunctional derivatives are also particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In some embodiments, a strong nucleophile (including but not limited tohydroxylamine) can be reacted with an carbonyl group, including a ketonegroup present in a non-natural amino acid to form an oxime in thepresence of an accelerant described herein (although such a reaction maybe less efficient in the absence of an accelerant described herein); thesubsequent oxime group in some cases can be further reduced by treatmentwith an appropriate reducing agent. Alternatively, the strongnucleophile can be incorporated into the polypeptide via a non-naturalamino acid and used to react preferentially with a carbonyl group,including a ketone group present in the water soluble polymer to form anoxime in the presence of an accelerant described herein (although such areaction may be less efficient in the absence of an accelerant describedherein). Generally, at least one terminus of the PEG molecule isavailable for reaction with the non-natural amino acid.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is used in branched formsthat can be prepared by addition of ethylene oxide to various polyols,such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(—PEG-OH)_(m) in which R is derived from a core moiety,such as glycerol, glycerol oligomers, or pentaerythritol, and mrepresents the number of arms. Multi-armed PEG molecules, such as thosedescribed in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490; 4,289,872;U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259, each ofwhich is incorporated by reference herein in its entirety, can also beused as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group, n is 100-1,000 (i.e.,average molecular weight is between about 5 kDa to about 40 kDa), and Zis an activated terminal group linked to CH by a chain of atoms ofdefined length. Yet another branched form, the pendant PEG, has reactivegroups, such as carboxyl, along the PEG backbone rather than at the endof PEG chains.

In order to maximize the desired properties of PEG, the total molecularweight and hydration state of the PEG polymer or polymers attached tothe biologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule.

The methods and compostions described herein may be used to producesubstantially homogenous preparations of polymer:protein conjugates.“Substantially homogenous” as used herein means that polymer:proteinconjugate molecules are observed to be greater than half of the totalprotein. The polymer:protein conjugate has biological activity and thepresent “substantially homogenous” PEGylated polypeptide preparationsprovided herein are those which are homogenous enough to display theadvantages of a homogenous preparation, e.g., ease m clinicalapplication in predictability of lot to lot pharmacokinetics.

As used herein, and when contemplating hydrophilicpolymer:polypeptide/protein conjugates, the term “therapeuticallyeffective amount” refers to an amount which provides benefit to apatient. The amount will vary from one individual to another and willdepend upon a number of factors, including the overall physicalcondition of the patient and the underlying cause of the disease orcondition. A therapeutically effective amount of the presentcompositions may be readily ascertained by one of ordinary skill in theart using publicly available materials and procedures. By way of exampleonly, a therapeutically effective amount may be an amount which gives anincrease in hematocrit for anemia patients; it may be an amount whichdecreases tumor size in cancer patients; it may be an amount whichincreases insulin levels in diabetic patients; or it may be an amountwhich decreases pain in patients suffering form chronic pain.

The number of water soluble polymers linked to a (modified) non-naturalamino acid polypeptide (i.e., the extent of PEGylation or glycosylation)described herein can be adjusted to provide an altered (including butnot limited to, increased or decreased) pharmacologic, pharmacokineticor pharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of the polypeptide is increased at leastabout 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, two fold, five-fold,10-fold, 50-fold, or at least about 100-fold over an unmodifiedpolypeptide.

In one embodiment, a polypeptide comprising a carbonyl- ordicarbonyl-containing non-natural amino acid is modified, in thepresence of an accelerant, with a PEG derivative that contains aterminal hydroxylamine moiety that is linked directly to the PEGbackbone, thus forming an oxime bond (although such a reaction may beless efficient in the absence of an accelerant described herein). Insome embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂

where R is a simple allyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between about 5 kDa toabout 40 kDa). The molecular weight of the polymer may be of a widerange, including but not limited to, between about 100 Da and about100,000 Da or more. The molecular weight of the polymer may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of the polymeris between about 100 Da and about 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and about 40,000Da. In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and about 40,000 Da. In some embodiments, the molecularweight of the polymer is between about 5,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of the polymer is between about10,000 Da and about 40,000 Da.

In another embodiment, a polypeptide comprising a carbonyl- ordicarbonyl-containing amino acid is modified with a PEG derivative thatcontains a terminal hydroxylamine moiety that is coupled to the PEGbackbone by means of an amide bond, thus forming an oxime bond (althoughsuch a reaction may be less efficient in the absence of an accelerantdescribed herein) further coupled to the PEG backbone by means of anamide bond. In some embodiments, the hydroxylamine-terminal PEGderivatives have the structure:

RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between about 5 kDa toabout 40 kDA).

In another embodiment, a polypeptide comprising a carbonyl- ordicarbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydroxylamine moiety thus forming anoxime bond (although such a reaction may be less efficient in theabsence of an accelerant described herein), with each chain of thebranched PEG having an average molecular weight ranging from about 10kDa to about 40 kDa and, in other embodiments, from about 5 kDa to about20 kDa. In some embodiments, the PEG derivatives containing ahydroxylamine group will have the structure:

[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000. Themolecular weight of the branched chain PEG may be between about 1,000 Daand about 100,000 Da, including but not limited to, 100,000 Da, 95,000Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da,60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da,7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000Da. In some embodiments, the molecular weight of the branched chain PEGis between about 1,000 Da and about 50,000 Da. In some embodiments, themolecular weight of the branched chain PEG is between about 1,000 Da andabout 40,000 Da. In some embodiments, the molecular weight of thebranched chain PEG is between about 5,000 Da and about 40,000 Da. Insome embodiments, the molecular weight of the branched chain PEG isbetween about 5,000 Da and about 20,000 Da.

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995). Methods for activationof polymers can also be found in WO 94/17039, U.S. Pat. No. 5,324,844,WO 94/18247, WO 94/04193, U.S. Pat. No. 5,219,564, U.S. Pat. No.5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698, and more WO 93/15189,and for conjugation between activated polymers and enzymes including butnot limited to Coagulation Factor VIII (WO 94/15625), haemoglobin (WO94/09027), oxygen carrying molecule (U.S. Pat. No. 4,412,989),ribonuclease and superoxide dismutase (Veronese at al., App. Biochem.Biotech. 11: 141-52 (1985)).

If necessary, the PEGylated non-natural amino acid polypeptidesdescribed herein obtained from the hydrophobic chromatography can bepurified further by one or more procedures known to those of ordinaryskill in the art including, but are not limited to, affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;reverse phase HPLC; gel filtration (using, including but not limited to,SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusionchromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), or extraction. Apparentmolecular weight may be estimated by GPC by comparison to globularprotein standards (Preneta A Z, PROTEIN PURIFICATION METHODS, APRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989, 293-306). Thepurity of the non-natural amino acid polypeptide:PEG conjugate can beassessed by proteolytic degradation (including but not limited to,trypsin cleavage) followed by mass spectrometry analysis. Pepinsky R B,et. al., J. Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

D. Use of Linking Groups and Applications, Including Polypeptide Dinersand Multimers

In addition to adding desired functionality directly to the non-naturalamino acid polypeptide, the non-natural amino acid portion of thepolypeptide may first be modified with a multifunctional (e.g., bi-,tri, tetra-) linker molecule that then subsequently is further modified.That is, at least one end of the multifunctional linker molecule reactswith at least one non-natural amino acid in a polypeptide and at leastone other end of the multifunctional linker is available for furtherfunctionalization. If all ends of the multifunctional linker areidentical, then (depending upon the stoichiometric conditions)homomultimers of the non-natural amino acid polypeptide may be formed.If the ends of the multifunctional linker have distinct chemicalreactivities, then at least one end of the multifunctional linker groupcan react to as to be bound to the non-natural amino acid polypeptideand the other end can subsequently react with a different functionality,including by way of example only, a desired functionality.

The multifunctional linker group has the general structure:

wherein:

each X is independently NH₂, —C(═O)R₉, —SR′ or -J-R, where R₉ is H orOR′, where

-   -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl; each R″ is independently H, alkyl, substituted        alkyl, or a protecting group, or when more than one R″ group is        present, two R″ optionally form a heterocycloalkyl;    -   each R′ is independently H, alkyl, or substituted alkyl;    -   each L is independently selected from the group consisting of        alkylene, substituted alkylene, alkenylene, substituted        alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,        —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1,        2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, -(alkylene or        substituted alkylene)NR′C(O)O-(alkylene or substituted        alkylene)-, —O—CON(R′)-(alkylene or substituted alkylene)-,        —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,        —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,        —N(R′)C(O)O-(alkylene or substituted alkylene)-,        —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)N(R′)-(alkylene or        substituted alkylene)-, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′);    -   L₁ is optional, and when present, is        —C(R′)_(p)—NR′—C(O)O-(alkylene or substituted alkylene)- where p        is 0, 1, or 2;    -   W is NH₂, —C(═O)R9, —SR′ or -J-R; and n is 1 to 3    -   provided that X and L-L₁-W together independently each provide        at least one of the following (a) a hydroxylamine group capable        of reacting with a carbonyl (including a dicarbonyl) group on a        non-natural amino acid or a (modified) non-natural amino acid        polypeptide; (b) a carbonyl group (including a dicarbonyl group)        capable of reacting with an hydroxylamine group on a non-natural        amino acid or a (modified) non-natural amino acid polypeptide;        or (c) a carbonyl group (including a dicarbonyl group) capable        of undergoing an exchange reaction with an oxime group on a        non-natural amino acid or a (modified) non-natural amino acid        polypeptide.

In a further or alternative illustrative embodiment, the molar ratio ofa compound of Formula (I) or Formula (XIV) to the multifunctional linkerof Formula (XIX) is about 1:2; 1:1; 1.5:1; 1.5:2; 2:1; 1:1.5; 2:1.5; or1.5 to 2.

A bifunctional homolinker in which the linker has two identical ends,i.e., hydroxylamine groups, can be used to form coupled polypeptides viathe formation of oxime bonds, wherein the formation of such coupledpolypeptides is performed in the presence of at least one accelerant(although the oxime bond may also occur at a slower reaction rate in theabsence of the accelerant). The use of an accelerant described herein inthe formation of polypeptide dimers and multimers is expected to providesignificant benefit because that stoichiometric ratio of the linker tothe first polypeptide, or the ratio of the linker-(first polypeptide)complex to the second polypeptide will be closer to stoichiometric inthe presence of the accelerant than in the absence of the accelerant(or, further, as the molar ratio of accelerant increases, the closer theratios of the aforementioned reactants will be to stoichiometric). Thestoichiometric ratio (or molar ratio) is an important factor in themodification of polypeptides because of the expense of the reagents(including the polypeptide and the molecules for conjugation) and thedifficulty in purification. Thus, the use of the accelerants providedherein can be used to reduce the cost and waste resulting from themodification of non-natural amino acid polypeptides, including theformation of polypeptide dimers or multimers, or the linking of anydesired group or functionality to a polypeptide.

Such a linker may be used to form a homodimer of a carbonyl- ordicarbonyl-containing non-natural amino acid polypeptide to form twooxime bonds either or both of which are formed in the presence of atleast one accelerant (although the oxime bond may also occur at a slowerreaction rate in the absence of the accelerant). Alternatively, if oneend of such a linker is protected, then such a partially protectedlinker can be used to bind the unprotected hydroxylamine end to acarbonyl- or dicarbonyl-containing non-natural amino acid polypeptidevia an oxime bond, leaving the other protected end available for furtherlinking reactions following deprotection. Alternatively, carefulmanipulation of the stoichiometry of the reagents may provide a similarresult (a heterodimer), albeit a result in which the desired heterodimerwill likely be contaminated with some homodimer.

Such a linker may also be used to form a homodimer of ahydroxylamine-containing non-natural amino acid polypeptide to form twooxime bonds either or both of which are formed in the presence of atleast one accelerant (although the oxime bond may also occur at a slowerreaction rate in the absence of the accelerant). Alternatively, if oneend of such a linker is protected, then such a partially protectedlinker can be used to bind the unprotected carbonyl end to ahydroxylamine-containing non-natural amino acid polypeptide via an oximebond, leaving the other protected end available for further linkingreactions following deprotection. Alternatively, careful manipulation ofthe stoichiometry of the reagents may provide a similar result (aheterodimer), albeit a result in which the desired heterodimer willlikely be contaminated with some homodimer.

A multifunctional heterolinkers in which each linker has more than onetype of terminal reactive group, i.e., hydroxylamine, oxime andthioester groups, can be used to form coupled polypeptides via theformation of at least one oxime bond, wherein the formation of the oximebond is performed in the presence of at least one accelerant. Such alinker may be used to form a heterodimer of a non-natural amino acidpolypeptide using the accelerant-promoted oxime-based chemistrydiscussed throughout this specification.

The methods and compositions described herein also provide forpolypeptide combinations, such as homodimers, heterodimers,homomultimers, or heteromultimers (i.e., trimers, tetramers, etc.). Byway of example only, the following description focuses on the GHsupergene family members, however, the methods, techniques andcompositions described in this section can be applied to virtually anyother polypeptide which can provide benefit in the form of dimers andmultimers, including by way of example only: alpha-1 antitrypsin,angiostatin, antihemolytic factor, antibody, antibody fragments,apolipoprotein, apoprotein, atrial natriuretic factor, atrialnatriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765, NAP-2,ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG,calcitonin, c-kit ligand, cytokine, CC chemokine, monocytechemoattractant protein-1, monocyte chemoattractant protein-2, monocytechemoattractant protein-3, monocyte inflammatory protein-1 alpha,monocyte inflammatory protein-1beta, RANTES, 1309, R83915, R91733, HCC1,T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen,colony stimulating factor (CSF), complement factor 5a, complementinhibitor, complement receptor 1, cytokine, epithelial neutrophilactivating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF),epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, anyinterferon-like molecule or member of the IFN family, interleukin (IL),IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,FLT, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TGF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone. The non-natural amino acid polypeptide may also behomologous to any polypeptide member of the growth hormone supergenefamily.

Thus, encompassed within the methods, techniques and compositionsdescribed herein are a GH supergene family member polypeptide containingone or more non-natural amino acids bound to another GH supergene familymember or variant thereof or any other polypeptide that is a non-GHsupergene family member or variant thereof, either directly to thepolypeptide backbone or via a linker. Due to its increased molecularweight compared to monomers, the GH supergene family member dimer ormultimer conjugates may exhibit new or desirable properties, includingbut not limited to different pharmacological, pharmacokinetic,pharmacodynamic, modulated therapeutic half-life, or modulated plasmahalf-life relative to the monomeric GH supergene family member. In someembodiments, the GH supergene family member dimers described herein willmodulate the dimerization of the GH supergene family member receptor. Inother embodiments, the GH supergene family member dimers or multimersdescribed herein will act as a GH supergene family member receptorantagonist, agonist, or modulator.

In some embodiments, the methods and compositions described hereinprovide multimers comprising one or more GH supergene family memberformed by reactions with water soluble activated polymers that have thestructure:

R—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X

wherein n is from about 5 to 3,000, m is 2-10, X can be a hydroxylamineor carbonyl- or dicarbonyl-containing moiety, and R is a capping group,a functional group, or a leaving group that can be the same or differentas X. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.

Using the chemistry detailed throughout this specification, one ofordinary skill in the art could design a linker in which at least onefunctional group can form an oxime group, in the presence of theaccelerant disclosed herein, with a non-natural amino acid polypeptide;the other functional groups on the linker could utilize other knownchemistry, including the nucleophile/electrophile based chemistry wellknown in the art of organic chemistry.

The formation of a polypeptide dimer or multimer linked together via atleast one oxime group from (a) the reaction of ahydroxylamine-containing non-natural amino acid polypeptide and acarbonyl-containing reagent, or (b) the reaction of acarbonyl-containing non-natural amino acid polypeptide and ahydroxylamine-containing reagent, can be enhanced by addition of anaccelerant to the reaction mixture. Such an accelerant is a compoundthat has at least one of the following properties: (a) increase the rateof reaction between a carbonyl-containing compound and ahydroxylamine-containing compound to form an oxime-containing compound,where the increase in rate is relative to the reaction in the absence ofthe accelerant; (b) lower the activation energy of the reaction betweena carbonyl-containing compound and a hydroxylamine-containing compoundto form an oxime-containing compound, where the decrease in activationenergy is relative to the reaction in the absence of the accelerant; (c)increase the yield of an oxime-containing compound from the reaction ofa carbonyl-containing compound with a hydroxylamine-containing compound,where the increase in yield is relative to the reaction in the absenceof the accelerant; (d) lower the temperature at which acarbonyl-containing compound reacts with a hydroxylamine-containingcompound to form an oxime-containing compound, where the decrease intemperature is relative to the reaction in the absence of theaccelerant; (e) decrease the time necessary to react acarbonyl-containing compound with a hydroxylamine-containing compound toform an oxime-containing compound, wherein the decrease in time isrelative to the reaction in the absence of accelerant; (f) decrease theamount of reagents necessary to form an oxime group on a non-naturalamino acid polypeptide, wherein the decrease in amount of reagents isrelative to the reaction in the absence of accelerant; (g) decrease theside products resulting from the reaction of a carbonyl-containingcompound with a hydroxylamine-containing compound to form anoxime-containing compound, wherein the decrease in side products isrelative to the reaction in the absence of accelerant; (h) does notirreversibly destroy the tertiary structure of a polypeptide undergoingan oxime-forming reaction in the presence of an accelerant (excepting,of course, where the purpose of the reaction is to destroy such tertiarystructure); (i) can be separated from an oxime-containing compound invacuo; and (j) modulate the reaction of a carbonyl-containing compoundwith a hydroxylamine-containing compound. In further embodiments, theaccelerant has at least two of the aforementioned properties, three ofthe aforementioned properties, four of the aforementioned properties,five of the aforementioned properties, six of the aforementionedproperties, seven of the aforementioned properties, eight of theaforementioned properties, nine of the aforementioned properties, or allof the aforementioned properties. In a further embodiment, theaccelerant has none of the aforementioned properties.

The use of an accelerant includes the use of a single accelerant ormultiple accelerants. In addition, the molar ratio of accelerant tocarbonyl-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the molar ratio of accelerant tohydroxylamine-containing compound includes values between about 0.5:1 to5000:1, including by way of example only 4000:1, 3000:1, 2000:1, 1000:1,500:1, 400:1, 300:1, 200:1, 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1,8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, and0.5:1. Further, the accelerant includes compounds that can besubstantially removed in vacuo from the resulting oxime-containingcompound. Further, the accelerant includes compounds containing adiamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazidemoiety.

Further, in any of the aforementioned aspects or embodiments, theaccelerant is selected from the group consisting of bifunctionalaromatic amines, oxoamine derivatives, and compounds having thefollowing structures:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), C(═NH)—NH and SO, SO₂.

In a further embodiment, the accelerant is a bifunctional aromaticamine. In a further embodiment, the aromatic amine is selected from thegroup:

Bifunctional Aromatic Amines:

In a further embodiment, the accelerant is an oxoamine derivative. In afurther embodiment, the oxoamine derivative is selected from the group:

Oxoamine Derivatives:

Further, the accelerant include compounds selected from the groupconsisting of:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), and C(—NH)—N—H. Further, in any of theaforementioned aspects or embodiments, the accelerant is selected fromthe compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by wayof example any of compounds 6, 8, 10, 7, and 20 of FIG. 5. In any of theaforementioned aspects or embodiments, the accelerant includes an agentthat can form a hydrazone upon reaction with a carbonyl-containinggroup. Further, in any of the aforementioned aspects the accelerantactivity depends on the rate of reaction with the ketone moiety and thestability of the resulting intermediate. Further, in any of theaforementioned aspects or embodiments, the pH of the reaction mixturecomprising the accelerant, the carbonyl-containing compound and thehydroxylamine-containing compound is between about 2.0 and 10; betweenabout 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0;between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; betweenabout 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0;between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0;about 5.5; about 6.0; about 6.5; and about 7.0.

Expression in Alternate Systems

Several strategies have been employed to introduce non-natural aminoacids into proteins in non-recombinant host cells, mutagenized hostcells, or in cell-free systems. These systems are also suitable for usein making the non-natural amino acid polypeptides described herein.Derivatization of amino acids with reactive side-chains such as Lys, Cysand Tyr resulted in the conversion of lysine to N²-acetyl-lysine.Chemical synthesis also provides a straightforward method to incorporatenon-natural amino acids. With the recent development of enzymaticligation and native chemical ligation of peptide fragments, it ispossible to make larger proteins. See, e.g., P. E. Dawson and S. B. H.Kent, Annu. Rev. Biochem, 69:923 (2000). Chemical peptide ligation andnative chemical ligation are described in U.S. Pat. No. 6,184,344, U.S.Patent Publication No. 2004/0138412, U.S. Patent Publication No.2003/0208046, WO 02/098902, and WO 03/042235, which are incorporated byreference herein. A general in vitro biosynthetic method in which asuppressor tRNA chemically acylated with the desired non-natural aminoacid is added to an in vitro extract capable of supporting proteinbiosynthesis, has been used to site-specifically incorporate over 100non-natural amino acids into a variety of proteins of virtually anysize. See, e.g., V. W. Cornish, D. Mendel and P. G. Schultz, Angew.Chem. Int. Ed. Engl., 1995, 34:621 (1995); C. J. Noren, S. J.Anthony-Cahill, M. C. Griffith, P. G. Schultz, A general method forsite-specific incorporation of non-natural amino acids into proteins,Science 244:182-188 (1989); and, J. D. Bain, C. G. Glabe, T. A. Dix, A.R. Chamberlin, E. S. Diala, Biosynthetic site-specific incorporation ofa non-natural amino acid into a polypeptide, J. Am. Chem. Soc.111:8013-8014 (1989). A broad range of functional groups has beenintroduced into proteins for studies of protein stability, proteinfolding, enzyme mechanism, and signal transduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the non-natural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe non-natural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol. 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskom, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karubrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the non-naturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Er-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaka, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS, Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate non-natural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing non-natural aminoacids. For example, see the following publications and references citedwithin, which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am.

Chem, 88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit. RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with Non-naturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety ofnon-natural side chains, including cofactors, spin labels andoligonucleotides into proteins in vitro. See, e.g., Corey, D. R.,Schultz, P. G. Generation of a hybrid sequence-specific single-strandeddeoxyribonuclease, Science, 238(4832):1401-1403 (1987); Kaiser, E. T.,Lawrence D. S., Rokita, S. E. The chemical modification of enzymaticspecificity, Annu Rev Biochem, 54:565-595 (1985); Kaiser, E. T.,Lawrence, D. S. Chemical mutation of enzyme active sites, Science,226(4674):505-511 (1984); Neet, K. E., Nanci A, Koshland, D. E.Properties of thiol-subtilisin, J Biol. Chem, 243(24):6392-6401 (1968);Polgar, L. et M. L. Bender. A new enzyme containing a syntheticallyformed active site. Thiol-subtilisin. J. Am. Chem Soc, 88:3153-3154(1966); and, Pollack, S. J., Nakayama, G. Schultz, P. G. Introduction ofnucleophiles and spectroscopic probes into antibody combining sites,Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem 62:483-514 (1993); and, Krieg, U. C., Walter,P., Hohnson, A. E. Photocrosslinking of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the signal recognitionparticle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that non-natural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of non-natural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing non-natural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

The following patents are incorporated by reference in their entiretyfor in vivo methods for incorporating non-natural amino acids intoproteins and other polypeptides, and for methods for producing theappropriate synthetases/tRNAs: U.S. Pat. Nos. 7,045,337 and 7,083,970.

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an non-natural aminoacid. Conventional site-directed mutagenesis was used to introduce thestop codon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagensis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the non-natural amino acid was incorporated in response to theUAG codon which gave a protein containing that amino acid at thespecified position. Experiments using [³H]-Phe and experiments withα-hydroxy acids demonstrated that only the desired amino acid isincorporated at the position specified by the UAG codon and that thisamino acid is not incorporated at any other site in the protein. See,e.g., Noren, et al, supra; Kobayashi et al., (2003) Nature StructuralBiology 10(6):425-432; and, Elrman, J. A., Mendel, D., Schultz, P. G.Site-specific incorporation of novel backbone structures into proteins,Science, 255(5041):197-200 (1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded potential catalysis tovarious chemical reactions. Studies have identified RNA molecules thatcan catalyze aminoacyl-RNA bonds on their own (2′)3′-termini(Illangakekare et al., 1995 Science 267:643-647), and an RNA moleculewhich can transfer an amino acid from one RNA molecule to another (Lohseet al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturalamino acids. Substrate-immobilized forms of enzymatic molecules that canaminoacylate tRNAs, including but not limited to, ribozymes, may enableefficient affinity purification of the aminoacylated products. Examplesof suitable substrates include agarose, sepharose, and magnetic beads.The production and use of a substrate-immobilized form of ribozyme foraminoacylation is described in Chemistry and Biology 2003, 10:1077-1084and U.S. Patent Application Publication 2003/0228593, which isincorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.,Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), toavoid the use of synthetases in aminoacylation. Such methods or otherchemical aminoacylation methods may be used to aminoacylate tRNAmolecules described herein.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(Asn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Aminoacylate tRNAs ribozymes can be immobilized on a substrate so as toenable efficient affinity purification of the aminoacylated tRNAs.Examples of suitable substrates include, but are not limited to,agarose, sepharose, and magnetic beads. Ribozymes can be immobilized onresins by taking advantage of the chemical structure of RNA, such as the3′-cis-diol on the ribose of RNA can be oxidized with periodate to yieldthe corresponding dialdehyde to facilitate immobilization of the RNA onthe resin. Various types of resins can be used including inexpensivehydrazide resins wherein reductive amination makes the interactionbetween the resin and the ribozyme an irreversible linkage. Synthesis ofaminoacyl-tRNAs can be significantly facilitated by this on-columnaminoacylation technique. Kourouklis et al. Methods 2005; 36:239-4describe a column-based aminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAsdescribed herein may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transaction/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Stephan in Scientist 2005 Oct. 10; pages 30-33 describes additionalmethods to incorporate non-natural amino acids into proteins. Lu et al.in Mol. Cell. 2001 October; 8(4):759-69 describe a method in which aprotein is chemically ligated to a synthetic peptide containingnon-natural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate non-naturalamino acids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired non-natural amino acid.The translational machinery of the oocyte then inserts the non-naturalamino acid at the position specified by UAG. This method has allowed invivo structure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g. P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate non-natural amino acids directly intoproteins in vivo offers a wide variety of advantages, including by wayof example only, high yields of mutant proteins, technical ease, thepotential to study the mutant proteins in cells or possibly in livingorganisms and the use of these mutant proteins in therapeutic treatmentsand diagnostic uses. The ability to include non-natural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci. 7:419 (1998).

It may also be possible to obtain expression of a non-natural amino acidpolypeptide described herein using a cell-free (in-vitro) translationalsystem. Translation systems may be cellular or cell-free, and may beprokaryotic or eukaryotic. Cellular translation systems include, but arenot limited to, whole cell preparations such as permeabilized cells orcell cultures wherein a desired nucleic acid sequence can be transcribedto mRNA and the mRNA translated. Cell-free translation systems arecommercially available and many different types and systems arewell-known. Examples of cell-free systems include, but are not limitedto, prokaryotic lysates such as Escherichia coli lysates, and eukaryoticlysates such as wheat germ extracts, insect cell lysates, rabbitreticulocyte lysates, rabbit oocyte lysates and human cell lysates.Eukaryotic extracts or lysates may be preferred when the resultingprotein is glycosylated, phosphorylated or otherwise modified becausemany such modifications are only possible in eukaryotic systems. Some ofthese extracts and lysates are available commercially (Promega; Madison,Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.;GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, such as the caninepancreatic extracts containing microsomal membranes, are also availablewhich are useful for translating secretory proteins. In these systems,which can include either mRNA as a template (in-vitro translation) orDNA as a template (combined in-vitro transcription and translation), thein vitro synthesis is directed by the ribosomes. Considerable effort hasbeen applied to the development of cell-free protein expression systems.See, e.g., Kim, D. M. and J. R. Swartz, Biotechnology andBioengineering, 74:309-316 (2001); Kim, D. M. and J. R. Swartz,Biotechnology Letters, 22, 1537-1542, (2000); Kim, D. M., and J. R.Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D. M., and J.R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999); andPatnaik, R. and J. R. Swartz, Biotechniques 24, 862-868, (1998); U.S.Pat. No. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO00/55353; WO 90/05785, which are incorporated by reference herein.Another approach that may be applied to the expression of non-naturalamino acid polypeptides includes the mRNA-peptide fusion technique. See,e.g., R. Roberts and J. Szostak, Proc. Natl. Acad. Sci. (USA)94:12297-12302 (1997); A. Frankel, et al., Chemistry & Biology10:1043-1050 (2003). In this approach, an mRNA template linked topuromycin is translated into peptide on the ribosome. If one or moretRNA molecules has been modified, non-natural amino acids can beincorporated into the peptide as well. After the last mRNA codon hasbeen read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of non-natural aminoacid polypeptides comprising one or more non-natural amino acids toidentify polypeptides having desired properties. More recently, in vitroribosome translations with purified components have been reported thatpermit the synthesis of peptides substituted with non-natural aminoacids. See, e.g., A. Forster et al., Proc. Natl Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

EXAMPLES Example 1 Improved Oxime Formation in the Presence ofAccelerant

A carbonyl-containing compound (non-natural amino acid polypeptide) isshown in Scheme 1A. The keto group of the amino acidpara-acetylphenylalanine incorporated into the protein reacts with ahydroxylamine-containing compound to form a relatively stable oxime.This reaction is highly specific. A faster reaction was observed in thepresence of an accelerant (Scheme 1B).

Example 2 Screening of Potential Accelerants Using 30 K PEG hGH-pAcFConjugation

Human growth hormone (hGH) with para-acetylphenylalanine substituted fortyrosine at position 35 was used to screen a panel of 20 compounds (FIG.5). hGH was buffer exchanged into hGH reaction buffer (20 mM NaOAc, 20mg/ml glycine, 5 mg/ml Mannitol, 1 mM EDTA, pH 4.0) using a PD 10column, and concentrated to 10 mg/ml using a Centrocon (10 K MWCO)concentrator. hGH (10 μl) was mixed with 3.6 μl mono hydroxylamine 30 KPEG (2.5 mM), 3 μl of potential accelerant solution (200 mM in hGHreaction buffer) and buffer to give a final volume of 30 μl. The molarratio of hGH:PEG was 1:2. The reaction mixtures were incubated at 28° C.for 16 hours and 36 hours and analyzed by SDS-PAGE (FIG. 6). Each laneof the gels shown in FIG. 6 is labeled with the compound tested as shownin FIG. 5. The last lane in each gel was a control reaction (noaccelerant). Compounds 6, 7, 8, 10, and 20 were accelerants. Twocompounds from the panel, compounds 7 and 20 (acetic hydrazide)(pictured in FIG. 5), were accelerants and were further evaluated undersimilar reaction conditions with a higher hGH protein concentration (8mg/ml). The reaction mixtures were incubated at 28° C. for 16 hours, andthe results from SDS-PAGE analysis are shown in FIG. 7. Lane 1 was areaction mixture with a hGH:PEG molar ratio of 1:2 and 50 mM ofaccelerant compound 7. Lane 2 was a reaction mixture with a hGH:PEGmolar ratio of 1:2 and 50 mM of accelerant compound 20. Lane 3 was areaction mixture with a hGH:PEG molar ratio of 1:2 with no accelerant.Lane 4 was a reaction mixture with a hGH:PEG molar ratio of 1:5 with noaccelerant. After 16 hours, both compounds were shown to catalyze thereaction.

Example 3 LCMS Analysis of hGH after Incubation with Accelerant

In hGH reaction buffer, wild type hGH (5.8 mg/ml) was incubated withvarious concentrations of the accelerant acetic hydrazide (200 mM, 100mM, 50 mM, 25 mM, 12.5 mM, 6.25 n3M and 0 mM) at 28° C. for 48 hours.The accelerant was removed through dialysis (10 k MWCO). The resultingprotein solutions were analyzed by LCMS (FIG. 8).] FIG. 10A shows thetotal LCMS trace. FIG. 8B shows the mass spectrum of hGH withoutaccelerant. FIG. 8C shows the mass spectrum of hGH with 200 mMaccelerant acetic hydrazide.

An accelerant for protein conjugation preferably does not exert anydeteriorative effects on protein, such as fragmentation, precipitation,and undesired covalent modification. No fragmentation was observed whenusing accelerants 7 and 20 (shown in FIG. 5) based on SDS-PAGE analysisand protein precipitation in all the conditions used with both scFv andhGH. For example, no covalent modification could be detected with LCMSafter 48 hour of incubation of wild type hGH with up to 200 mMaccelerant 20 (acetic hydrazide). The measured molecular weights of hGHin all the conditions are same and match theoretical value 22256.

Example 4 One Step Dimerization of scFv-pAcF

Single chain Fv (scFv) 108 protein with para-acetylphenylalaninesubstituted at position 259 (scFv 108 259-pAcF) was used the followingconjugation experiment. scFv 108 259-pAcF in storage buffer was bufferexchanged into reaction buffer (150 mM NaCl, 20 mM NaOAc, 5 mM EDTA, pH4.0) using a PD 10 column. The protein solution was concentrated to 0.5mM, mixed with hydroxylamine homobifunctional 2 K PEG linker 2.5 mMstock solution and supplemented with acetic hydrazide as accelerant. Thefinal reaction mixture consisted of 147 μM homobifunctional 2 K PEGlinker, the corresponding concentration of pAcF-substituted scFv and 47mM acetic hydrazide. The reaction mixtures were incubated at 28° C. andanalyzed at different time points (6 hours, 20 hours, 44 hours) bySDS-PAGE (FIG. 2).

To make the one-step reaction more efficient and more practical with thehomobifunctional 2 K PEG linker, accelerants were used to facilitate thedimerization process. Without accelerant, very little dimer productcould be detected after 20 hours by SDS-PAGE. Lane 4 of the 6 hour, 20hour, and 44 hour gels show reaction mixtures with a scFv:PEG linkermolar ratio of 2.0:1 without the accelerant acetic hydrazide. On theother hand, in the presence of 47 mM acetic hydrazide, the dimer productappeared after 6 hours. As shown in FIG. 2, different protein and linkermolar ratios, 1.6:1, 2.0:1 and 2.4:1, were tested to scan for the bestconjugation conditions. Lane 1 of the 6 hour, 20 hour, and 44 hour gelsshow reaction mixtures with a scFv:PEG linker molar ratio of 1.6:1 withacetic hydrazide. Lane 2 of the 6 hour, 20 hour, and 44 hour gels showreaction mixtures with a scFv:linker molar ratio of 2.0:1 with acetichydrazide. Lane 3 of the 6 hour, 20 hour, and 44 hour gels show reactionmixtures with a scFv:linker molar ratio of 2.4:1 with acetic hydrazide.As a control, scFv 108 was incubated with 47 mM acetic hydrazide in theabsence of the homobifunctional PEG linker for 44 hours. No dimerformation was observed. Lane 5 of the 6 hour, 20 hour, and 44 hours gelsshow reaction mixtures of scFv and accelerant acetic hydrazide withoutthe PEG linker. This result indicates that the accelerant, acetichydrazide, does not facilitate the formation of intermolecular disulfidebonds among scFv. In the presence of homobifunctional PEG linker, thedimer is produced through the oxime formation between the PEG linker andprotein.

Example 5 Mono Hydroxylamine 30 K PEG and scFv-pAcF Conjugation

scFv 108 259-pAcF 0.5 mM stock solution in reaction buffer mentionedabove (10 μl) is mixed with various amounts of mono hydroxylamine 30 KPEG 2.5 mM stock solution, 2.2 μl of 200 mM acetic hydrazide stocksolution and reaction buffer. The reaction mixtures with a finalreaction volume of 22 μl have different molar ratios of scFv:PEG (1:3 or1:5). The accelerating effect of acetic hydrazide on conjugation of monohydroxylamine 30 K PEG and scFv was evaluated at protein and PEG molarratio of 1:3 and 1:5 with and without accelerant. The reaction mixtureswere incubated at 28° C. and were analyzed at different time points (20hours, 44 hours) by SDS-PAGE (FIG. 3). Lanes 1, 2, and 3 show 100%, 20%,and 10% of the starting scFv-pAcF, respectively. Lane 4 of the hour and44 hour gels show reaction mixtures with a scFv:PEG molar ratio of 1:3with 20 mM acetic hydrazide. Lane 5 of the 20 hour and 44 hour gels showreaction mixtures with a scFv:PEG molar ratio of 1:3 without accelerant.Lane 6 of the 20 hour and 44 hour gels show reaction mixtures with ascFv:PEG molar ratio of 1:5 with 20 mM acetic hydrazide. Lane 7 of the20 hour and 44 hour gels show reaction mixtures with a scFv:PEG molarratio of 1:5 without accelerant. The conjugation results demonstratedthat acetic hydrazide accelerates the conjugation reaction. The reactionat a 1:3 molar ratio of protein:PEG with accelerant proceeded fasterthan the reaction at a 1:5 ratio of protein:PEG without accelerant.

Similarly, the relative conjugation efficiency of differentconcentrations of the accelerant acetic hydrazide (5 mM, 20 mM, 80 mM)with a scFv:30 K PEG mono hydroxylamine molar ratio of 1:2 wereevaluated (FIG. 4). The reaction mixtures were incubated at 28° C. Lane1 shows the reaction mixture with 5 mM acetic hydrazide (scFv:30 K PEGmono hydroxylamine molar ratio of 1:2). Lane 2 shows the reactionmixture with 20 mM acetic hydrazide (scFv:30 K PEG mono hydroxylaminemolar ratio of 1:2). Lane 3 shows the reaction mixture with 80 mM acetichydrazide (scFv:30 K PEG mono hydroxylamine molar ratio of 1:2). Lane 4shows the reaction mixture with no acetic hydrazide (scFv:30 K PEG monohydroxylamine molar ratio of 1:5). Lanes 5, 6, and 7 show 10%, 20%, and100% of the starting scFv-pAcF, respectively. SDS-PAGE analysis showedthat the higher concentration of accelerant, the faster the conjugation.The conjugation at a scFv:PEG molar ratio of 1:2 in the presence of 80mM accelerant is faster than the conjugation at a scFv:PEG molar ratioof 1:5 without the accelerant.

Example 6 Small Molecule Studies of Accelerated Oxime Formation

Acetophenone (0.5 mM) was reacted with ethylhydroxylamine (1 mM) inaqueous solution buffered to a pH of about 4.0; a series of accelerants(20 mM) was added to this reaction mixture to determine the effect ofaccelerant identity on the rate and yield of oxime formation (see FIG.10( a)). The accelerants that were tested in this model reaction arepresented in FIG. 10( b). Aliquots were taken from the reaction mixtureand analyzed by high-performance liquid chromatography after 2 h, 5 h, 9h, and 24 h. In addition, for each of these samples, the ketone peakabsorbance was compared to the oxime peak absorbance at 260 nm usingUV/Vis spectoscopy. FIG. 11 presents the results after 2 h and 9 h ofreaction. As can be seen, all accelerants increase the rate of oximeformation; however, accelerants 1 and 7 (shown in FIG. 10( b)) providethe highest yield, with accelerant 1 providing the highest yieldfollowing longer reaction times. Without being bound to a particulartheory, the activity of the accelerant appears to depend on both therate of reaction with the ketone and on the stability of the hydrazoneintermediate.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons of ordinary skill in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

1. A reaction mixture comprising a compound comprising an aromaticketone moiety, a compound comprising a hydroxylamine moiety, and anaccelerant selecting from the group consisting of bifunctional aromaticamines, oxoamine derivatives, and compounds having the followingstructures:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), C(═NH)—NH, SO, and SO₂.
 2. The reaction mixture ofclaim 1, wherein the compound comprising an aromatic ketone moiety is anamino acid or a polypeptide.
 3. The reaction mixture of claim 1, whereinthe compound comprising an aromatic ketone moiety has the structure:

wherein: R is alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; R₁ is H, an amino protecting group, resin, amino acid,polypeptide, or polynucleotide; and R₂ is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide; wherein each R_(a) isindependently selected from the group consisting of H, halogen, alkyl,substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3,—C(O)N(R′)₂, —OR′, and —S(O)_(k)R′,
 4. The reaction mixture of claim 1,wherein the compound comprising a hydroxylamine moiety further comprisesa polymer moiety.
 5. The reaction mixture of claim 1, wherein thecompound comprising a hydroxylamine moiety has the structure:

wherein: each L is a linker independently selected from the groupconsisting of alkylene, substituted alkylene, alkenylene, substitutedalkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkyleneor substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substitutedalkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-,—C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, -(alkylene orsubstituted alkylene)NR′C(O)O-(alkylene or substituted alkylene)-,—O—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(O)O-(alkylene orsubstituted alkylene)-, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)N(R′)-(alkylene or substituted alkylene)-, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N—, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl.
 6. The reaction mixture ofclaim 1, wherein the accelerant is a bifunctional aromatic amine.
 7. Thereaction mixture of claim 1, wherein the accelerant is an oxoaminederivative.
 8. A method for derivatizing an amino acid of Formula (III),the method comprising contacting the amino acid with a reagent ofFormula (XXVII) in the presence of an accelerant, wherein Formula (III)corresponds to:

wherein: R is alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; R₁ is H, an amino protecting group, resin, amino acid,polypeptide, or polynucleotide; and R₂ is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide; wherein each R_(a) isindependently selected from the group consisting of H, halogen, alkyl,substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3,—C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, wherein Formula (XXVII) correspondsto:

wherein: each L is a linker independently selected from the groupconsisting of alkylene, substituted alkylene, alkenylene, substitutedalkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkyleneor substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substitutedalkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-,—C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, -(alkylene orsubstituted alkylene)NR′C(O)O-(alkylene or substituted alkylene)-,—O—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(O)O-(alkylene orsubstituted alkylene)-, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)N(R′)-(alkylene or substituted alkylene)-, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; and wherein the accelerantis selected from the group consisting of bifunctional aromatic amines,oxoamine derivatives, and compounds having the following structures:

wherein R_(x), R_(y) and R_(z) are selected from the group consistingof: L_(x)-H, L_(x)-alkyl, L_(x)-aryl, L_(x)-heteroaryl, L_(x)-alkenyl,L_(x)-alkynyl, L_(x)-alkoxy, and L_(x)-alkylamine, where L_(x) is abond, C(═O), C(═NH), C(═NH)—NH and SO, SO₂.
 9. The method of claim 8,wherein R is alkyl.
 10. The method of claim 8, wherein R is CH₃.
 11. Themethod of claim 8, wherein the accelerant has the structure:H₂N—NH—C(O)—R_(b), wherein R_(b) is alkyl, substituted alkyl, NH—NH₂, H,and alkoxy.
 12. The method of claim 11, wherein R_(b) is alkyl oralkoxy.
 13. The method of claim 8, wherein the accelerant is selectedfrom the group consisting of the compounds identified as 6, 7, 8, 10,and 20 of FIG.
 5. 14. The method of claim 8, wherein the accelerant is abifunctional aromatic amine.
 15. The method of claim 14, wherein thebifunctional aromatic amine is selected from the group consisting of:Bifunctional aromatic amines:


16. The method of claim 8, wherein the accelerant is an oxoaminederivative.
 17. The method of claim 16, wherein the oxoamine is selectedfrom the group consisting of: Oxoamine derivatives:


18. The method of claim 8, wherein the molecular weight of the PEG groupis between about 1,000 Da and about 40,000 Da.
 19. The method of claim8, wherein the amino acid is contacted with the reagent of Formula(XXVII) in aqueous solution at about room temperature.
 20. The method ofclaim 8, wherein the amino acid is contacted with the reagent of Formula(XXVII) in aqueous solution at a pH between about 4 to about
 10. 21. Themethod of claim 8, wherein the molar ratio of amino acid to the reagentof Formula (XXVII) is selected from the group of about 1:2; 1:1; 1.5:1;1.5:2; 2:1; 1:1.5; 2:1.5; and 1.5 to
 2. 22. The method of claim 8,wherein the amino acid of Formula (III) has been incorporatedsite-specifically during the in vivo translation of a polypeptide. 23.The method of claim 8, wherein the derivatized amino acid comprises atleast one oxime containing amino acid having the structure of Formula(XI-A):

wherein: R is H, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; R₁ is H, an amino protecting group, resin, amino acid,polypeptide, or polynucleotide; and R₂ is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide; wherein each R_(a) isindependently selected from the group consisting of H, halogen, alkyl,substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3,—C(O)N(R′)₂, —OR′, and —S(O)_(k)R1, R₅ is L-X, where X is a PEG; and Lis optional, and when present is a linker selected from the groupconsisting of alkylene, substituted alkylene, alkenylene, substitutedalkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkyleneor substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substitutedalkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-,—C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl.
 24. Thederivatized amino acid of claim
 23. 25. The derivatized amino acid ofclaim 24, wherein the amino acid is incorporated into a therapeuticprotein that is a member of the growth hormone supergene family.
 26. Thederivatized amino acid of claim 25, wherein the therapeutic protein ishuman growth hormone.